Determining the capability of a test compound to affect solid tumor stem cells

ABSTRACT

A small percentage of cells within an established solid tumor have the properties of stem cells. These solid tumor stem cells give rise both to more tumor stem cells and to the majority of cells in the tumor that have lost the capacity for extensive proliferation and the ability to give rise to new tumors. Thus, solid tumor heterogeneity reflects the presence of tumor cell progeny arising from a solid tumor stem cell. We have developed a xenograft model in which we have been able to establish tumors from primary tumors via injection of tumor cells in the mammary gland of severely immunodeficient mice. These xenograft assay have allowed us to do biological and molecular assays to characterize clonogenic solid tumor stem cells. We have also developed evidence that strongly implicates the Notch pathway, especially Notch 4, as playing a central pathway in carcinogenesis.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. application Ser. No.11/776,935, filed Jul. 12, 2007, now allowed, which is aContinuation-In-Part of U.S. application Ser. No. 11/529,869, filed Sep.29, 2006, now U.S. Pat. No. 7,850,961 which is a Continuation of U.S.application Ser. No. 10/343,692, filed Jan. 31, 2003, now U.S. Pat. No.7,115,360, which is a U.S. National Entry of International ApplicationPCT/US01/24243 filed Aug. 2, 2001, which in turn claims priority to:U.S. application Ser. No. 09/920,517 filed Aug. 1, 2001, now U.S. Pat.No. 6,984,522; U.S. Provisional Application 60/240,317 filed Oct. 13,2000; and U.S. Provisional Application 60/222,794, filed Aug. 3, 2000,all of which are herein incorporated by reference.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under CA075136 awardedby the National Institute of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

This invention relates to the diagnosis and treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer remains the number two cause of mortality in this country,resulting in over 500,000 deaths per year. Despite advances in detectionand treatment, cancer mortality remains high. Despite the remarkableprogress in understanding the molecular basis of cancer, this knowledgehas not yet been translated into effective therapeutic strategies.

In particular, breast cancer is the most common cancer in Americanwomen, with approximately one in nine women developing breast cancer intheir lifetime. Unfortunately, metastatic breast cancer is still anincurable disease. Most women with metastatic breast cancer succumb tothe disease.

Traditional modes of therapy (radiation therapy, chemotherapy, andhormonal therapy), while useful, have been limited by the emergence oftreatment-resistant cancer cells. Clearly, new approaches are needed toidentify targets for treating metastatic breast cancer and cancergenerally.

SUMMARY OF THE INVENTION

A small percentage of cells within an established solid tumor have theproperties of stem cells. These solid tumor stem cells give rise both tomore tumor stem cells and to the majority of cells in the tumor thathave lost the capacity for extensive proliferation and the ability togive rise to new tumors. Thus, solid tumor heterogeneity reflects thepresence of tumor cell progeny arising from a solid tumor stem cell.

In some embodiments, the present invention provides an isolated solidtumor stem cells, wherein: (a) the solid tumor stem cell is derived froma solid tumor; and (b) the solid tumor stem cell is tumorigenic.

In certain embodiments, the present invention provides an enrichedpopulation of solid tumor stem cells, wherein: (a) the tumor cells arederived from a solid tumor; (b) the solid tumor stem cells aretumorigenic; and (c) the solid tumor stem cell population is enriched atleast 2-fold relative to unfractionated tumor cells. In furtherembodiments, the enrichment is in the ability to form new tumorsrelative to unfractionated tumor cells. In particular embodiments, thepopulation is at least 5-fold, 10-fold, or at least 50-fold enriched.

In particular embodiments, the present invention provides a populationof cells that have been enriched for non-tumorigenic solid tumor cells,wherein: (a) the population is derived from a solid tumor; and (b) thepopulation is depleted for the ability to form tumors relative tounfractionated solid tumor cells.

In some embodiments, the present invention provides methods forenriching a population of cells for solid tumor stem cells, comprisingthe steps of: (a) dissociating a solid tumor to form a cell suspension;(b) contacting the dissociated cells with at least one reagent, whereinthe reagent either selectively binds to a solid tumor stem cell positivemarker or negative marker; and (c) selecting cells that bind to thereagent that selectively binds to a positive marker and/or that do notbind to the reagent that selectively binds to a negative marker, whereinthe selected cells are enriched in tumor stem cells as compared with theunfractionated population of solid tumor cells. In further embodiments,the reagent is an antibody or a lectin. In other embodiments, thereagent is conjugated to a fluorochrome or to magnetic particles. Incertain embodiments, the cell selection is performed is by flowcytometry, fluorescence activated cell sorting, panning, affinity columnseparation, and/or magnetic selection. In other embodiments, steps (b)and (c) comprise: (b) contacting the dissociated cells with acombination of reagents, wherein each reagent in the combination eitherselectively binds to either a solid tumor stem cell positive marker ornegative marker; and (c) selecting cells that bind to reagents thatselectively bind to the positive marker or that do not bind to reagentsthat selectively bind to the negative marker or a combination thereof,wherein the selected cells are enriched in tumor stem cells as comparedwith the population of unfractionated cells. In particular embodiments,the methods further comprise the step of: (d) isolating the selectedsolid tumor stem cells. In some embodiments, the methods furthercomprise the steps of: (d) introducing at least one selected cell to aculture medium that supports the growth of tumor stem cells; and (e)proliferating the selected cell in the culture medium. In particularembodiments, the methods further comprise the step of: (f) introducingthe proliferated cell into a host mammal. In yet other embodiments, themethods further comprise the steps of: (f) contacting the proliferatedcell with a test compound; and (g) determining the effect of the testcompound on the proliferated cell. In additional embodiments, themethods further comprise the steps of: (f) mixing a population ofnon-tumorigenic tumor cells with the solid tumor stem cells in culture,wherein the population of non-tumorigenic tumor cells (i) is derivedfrom a solid tumor; (ii) is depleted for the ability to form tumorsrelative to unfractionated solid tumor cells. In other embodiments, themethods further comprise the steps of: (g) transplanting the mixtureinto a host animal. In some embodiments, the methods further comprisethe steps of: (g) analyzing the mixture for an increase or decrease inthe ability of the solid tumor stem cells to survive or proliferate. Inparticular embodiments, the methods further comprise the step of: (f)isolating the proliferated solid tumor stem cell.

In some embodiments, the present invention provides methods forstimulating an immune response to a solid tumor stem cell, comprisingthe steps of: (a) obtaining an enriched population of solid tumor stemcells; wherein: (i) the tumor cells are derived from a solid tumor; (ii)the solid tumor stem cells are tumorigenic; and (iii) the solid tumorstem cell population is enriched at least 2-fold relative tounfractionated tumor cells; (b) treating the population to prevent cellreplication; and (c) administering the treated cell to a human or animalsubject in an amount effective for inducing an immune response to solidtumor stem cells. In certain embodiments, the treatment kills the solidtumor stem cells. In other embodiments, the administration is byinjection or by oral administration. In additional embodiments, themethods further comprise the step of (d) obtaining antibodies orantibody secreting hybridomas from the human or animal subject. Infurther embodiments, the methods further comprise the step of: (e)testing the obtained antibody for the ability to specifically bind tosolid tumor stem cells. In other embodiments, the methods furthercomprise the step of: (e) testing the obtained antibody for the abilityto bind to a polypeptide present on solid tumor stem cells. Inparticular embodiments, the methods further comprise the step of: (e)immunologically identifying a polypeptide present on solid tumor stemcells. In certain embodiments, the methods further comprise the step of:(e) immunologically identifying a polynucleotide encoding a polypeptidepresent on solid tumor stem cells. In further embodiments, the methodsfurther comprise the step of: (e) testing the obtained antibody for theability to reduce tumor growth.

In certain embodiments, the present invention provides methods forstimulating an immune response to a solid tumor stem cell, comprisingthe steps of: (a) obtaining an enriched population of solid tumor stemcells; wherein: (i) the tumor cells are derived from a solid tumor; (ii)the solid tumor stem cells are tumorigenic; and (iii) the solid tumorstem cell population is enriched at least 2-fold relative tounfractionated tumor cells; (b) mixing the tumor stem cells in an invitro culture with immune effector cells; (c) removing the immuneeffector cells from the culture; and (d) transplanting the immuneeffector cells into a host animal in a dose that is effective tostimulate an immune response in the animal.

In some embodiments, the present invention provides a purifiedpopulation of polynucleotides, wherein the polynucleotides have beenpurified from an enriched population of solid tumor stem cells, wherein:(a) the tumor cells are derived from a solid tumor; (b) the solid tumorstem cells are tumorigenic; and (c) the solid tumor stem cell populationis enriched at least at least 2-fold relative to unfractionated tumorcells. In certain embodiments, the polynucleotides are affixed to asolid surface. In further embodiments, the polynucleotides are affixedto the solid surface in an orderly array. In other embodiments, theorderly array is a microarray. In particular embodiments, thepolynucleotides are in a cDNA library. In some embodiments, thepolynucleotides have been amplified. In certain embodiments, thepolynucleotides are labeled. In additional embodiments, thepolynucleotides are used as a hybridization probe. In certainembodiments, the polynucleotides further comprise a microarray ofpolynucleotide sequences.

In additional embodiments, the present invention provides a purifiedpopulation of polypeptides, wherein the polypeptides have been purifiedfrom an enriched population of solid tumor stem cells, wherein: (a) thetumor cells are derived from a solid tumor; (b) the solid tumor stemcells are tumorigenic; and (c) the solid tumor stern cell population isenriched at least at least 2-fold relative to unfractionated tumorcells. In some embodiments, the polypeptides are used as a probe. Inparticular embodiments, the purified population of polypeptides furthercomprises a microarray of components, wherein the components areselected from the group consisting of cells, polynucleotides,polypeptides, and test compounds.

In some embodiments, the present invention provides method for analyzinga population of cells enriched for solid tumor stem cells for geneexpression patterns, comprising the steps of: (a) obtaining anpopulation of cells enriched for solid tumor stem cells; wherein: (i)the tumor cells are derived from a solid tumor; (ii) the solid tumorstem cells are tumorigenic; and (iii) the solid tumor stem cellpopulation is enriched at least 2-fold relative to unfractionated tumorcells; and (b) analyzing the population of cells for gene expressionpatterns. In certain embodiments, the analysis is by a method selectedfrom the group consisting of resequencing, high throughput screening,use of a microarray, use of analytical software for data collection andstorage, use of analytical software for flexible formatting of dataoutput, use of analytical software for statistical analysis ofindividual spot intensities to provide grouping and cluster analyses,and use of analytical software for linkage to external databases.

In certain embodiments, the present invention provides methods foranalyzing a population of cells enriched for solid tumor stem cells forprotein expression patterns, comprising the steps of: (a) obtaining anpopulation of cells enriched for solid tumor stem cells; wherein: (i)the tumor cells are derived from a solid tumor; (ii) the solid tumorstem cells are tumorigenic; and (iii) the solid tumor stem cellpopulation is enriched at least 2-fold relative to unfractionated tumorcells; and (b) analyzing the population of cells for protein expressionpatterns. In some embodiments, the analysis is by a method selected fromthe group consisting of mass spectrometry, high throughput screening,use of a microarray, use of analytical software for data collection andstorage, use of analytical software for flexible formatting of dataoutput, use of analytical software for statistical analysis ofindividual spot intensities to provide grouping and cluster analyses,and use of analytical software for linkage to external databases.

In additional embodiments, the present invention provides methods fordetermining the effect of a test compound on a solid tumor stem cell,comprising the steps of: (a) obtaining an enriched population of solidtumor stem cells, wherein: (i) the tumor cells are derived from a solidtumor; (ii) the solid tumor stem cells are tumorigenic; and (iii) thesolid tumor stem cell population is enriched at least 2-fold relative tounfractionated tumor cells; (b) contacting the obtained cells with thetest compound; and (c) determining the response of the contacted cellsto the test compound. In some embodiments, the solid tumor stem cellsare localized in a manner selected from the group consisting of inmonolayers in culture, in suspension in culture, and affixed to a solidsurface. In other embodiments, the contacting is effected at more thanone concentration of the test compound being tested. In particularembodiments, the contacting is effected using microfluidic methods. Infurther embodiments, the determination of the response of the contactedcells to the test compound comprises assaying for an effect selectedfrom the group consisting of tumor formation, tumor growth, tumor stemcell proliferation, tumor cell survival, tumor cell cycle status, andtumor stem cell survival. In certain embodiments, the methods furthercomprise the step of (d) identifying the target in the contacted cellswith which the test compound interacts.

In some embodiments, the present invention provides methods fordetermining the effect of a test compound on a solid tumor stem cell,comprising the steps of: (a) contacting a population of solid tumorcells with the test compound; (b) obtaining an enriched population ofsolid tumor stem cells from the contacted solid tumor cells, wherein:(i) the tumor cells are derived from a solid tumor; (ii) the solid tumorstem cells are tumorigenic; and (iii) the solid tumor stem cellpopulation is enriched at least 2-fold relative to unfractionated tumorcells; and (c) determining the response of the contacted cells to thetest compound. In certain embodiments, the contacting is effected usingmicrofluidic methods. In other embodiments, the determination of theresponse of the contacted cells to the test compound comprises assayingfor an effect selected from the group consisting of tumor formation,tumor growth, tumor stem cell proliferation, tumor cell survival, tumorcell cycle status, and tumor stem cell survival. In additionalembodiments, the test compound is in an array of other molecules. Inparticular embodiments, the methods further comprise the step of (d)identifying the target in the contacted cells with which the testcompound interacts.

In certain embodiments, the present invention provides methods fordetermining the effect of a test compound on a solid tumor stem cell,comprising the steps of: (a) obtaining an enriched population of solidtumor stem cells, wherein; (i) the tumor cells are derived from a solidtumor; (ii) the solid tumor stem cells are tumorigenic; and (iii) thesolid tumor stem cell population is enriched at least 2-fold relative tounfractionated tumor cells; (b) transplanting the obtained cells into ananimal; (c) administering a test compound to the animal; and (d)determining the response of the transplanted solid tumor stem cells tothe test compound. In particular embodiments, the animal is animmunocompromised animal (e.g., immunocompromised mouse selected fromthe group consisting of nude mouse, SCID mouse, NOD/SCID mouse,Beige/SCID mouse; and (32 microglobin deficient NOD/SCID mouse).

In further embodiments, the present invention provides methods fordetermining the effect of a test compound on a solid tumor stem cell,comprising the steps of: (a) administering a test compound to an animal;(b) obtaining from the animal an enriched population of solid tumor stemcells, wherein; (i) the tumor cells are derived from a solid tumor; (ii)the solid tumor stem cells are tumorigenic; and (iii) the solid tumorstem cell population is enriched at least 2-fold relative tounfractionated tumor cells; and (c) determining the response of thesolid tumor stem cells to the test compound. In particular embodiments,the animal is an immunocompromised animal (e.g., immunocompromised mouseselected from the group consisting of nude mouse, SCID mouse, NOD/SCIDmouse, Beige/SCID mouse; and β2 microglobin deficient NOD/SCID mouse).

In other embodiments, the present invention provides methods forscreening for a test compound that specifically binds to a solid tumorstem cell, comprising the steps of: (a) obtaining an enriched populationof solid tumor stem cells, wherein; (i) the tumor cells are derived froma solid tumor; (ii) the solid tumor stem cells are tumorigenic; and(iii) the solid tumor stem cell population is enriched at least 2-foldrelative to unfractionated tumor cells; (b) contacting the enrichedpopulation of solid tumor stem cells with a test compound underconditions suitable to allow complex formation; and (c) detectingcomplex formation between the test compound and a solid tumor stem cell,wherein the presence of the complex identifies the test compound asspecifically binding the tumor stem cell.

In other embodiments, the present invention provides methods forscreening for a test compound that specifically binds to a solid tumorstem cell, comprising the steps of: (a) contacting a population of solidtumor cells with a test compound under conditions suitable to allowcomplex formation; and (b) obtaining an enriched population of solidtumor stem cells from the contacted solid tumor cells, wherein; (i) thetumor cells are derived from a solid tumor; (ii) the solid tumor stemcells are tumorigenic; and (iii) the solid tumor stem cell population isenriched at least 2-fold relative to unfractionated tumor cells; (c)detecting complex formation between the test compound and a solid tumorstem cell, wherein the presence of the complex identifies the testcompound as specifically binding the tumor stem cell.

In some embodiments, the present invention provides methods fordiagnosing the presence of solid tumor stem cells, comprising the stepsof: (a) contacting the cells from a solid tumor with a reagent thatbinds to a positive marker for solid tumor stem cells; and (b) detectingthe contact between the reagent and the cells from the solid tumor,wherein an increased detection of the number of contacted cells ascompared with the number of contacted cells in a benign tumor identifiesthe tumor as containing solid tumor stem cells. In certain embodiments,the detection is by flow-cytometry or immunohistochemistry.

In other embodiments, the present invention provides an in vitro methodfor the proliferation of a tumor stem cells, comprising the steps of:(a) obtaining an enriched population of solid tumor stem cells, wherein;(i) the tumor cells are derived from a solid tumor; (ii) the solid tumorstem cells are tumorigenic; and (iii) the solid tumor stem cellpopulation is enriched at least 2-fold relative to unfractionated tumorcells; and (b) proliferating the obtained cells in a culture medium.

In further embodiments, the present invention provides an in vivo methodfor the proliferation of a tumor stem cells, comprising the steps of:(a) obtaining an enriched population of solid tumor stem cells, wherein;(i) the tumor cells are derived from a solid tumor; (ii) the solid tumorstem cells are tumorigenic; and (iii) the solid tumor stem cellpopulation is enriched at least 2-fold relative to unfractionated tumorcells; and (b) transplanting the isolated cell into a host mammal underconditions that allow the proliferation of solid tumor stem cells in thehost mammal. In additional embodiments, the methods further comprise thestep of: (c) isolating the proliferated tumor cells from the hostmammal.

In some embodiments, the present invention provides methods forproducing genetically modified tumor stem cells, comprising the stepsof: (a) obtaining an enriched population of solid tumor stem cells,wherein: (i) the tumor cells are derived from a solid tumor; (ii) thesolid tumor stem cells are tumorigenic; (iii) the solid tumor stem cellpopulation is enriched at least 2-fold relative to unfractionated tumorcells; (b) genetically modifying the obtained cells.

In other embodiments, the present invention provides methods forproducing genetically modified tumor stem cells, comprising the stepsof: (a) genetically modifying cells of a population of solid tumorcells; and (b) obtaining an enriched population of solid tumor stemcells from the genetically modified solid tumor cells, wherein: (i) thetumor cells are derived from a solid tumor; (ii) the solid tumor stemcells are tumorigenic; (iii) the solid tumor stem cell population isenriched at least 2-fold relative to unfractionated tumor cells.

In further embodiments, the genetic modification is performed in vitroor in vivo. In certain embodiments, the genetic modification is theintroduction of a plasmid into the solid tumor stem cell. In otherembodiments, the genetic modification is the introduction of a viralvector into the solid tumor stem cell. In some embodiments, the virushas been modified to express a protein that recognizes an antigen on thesolid tumor stem cell, thus specifically targeting the virus to solidtumor stem cell. In other embodiments, the methods further comprise thestep of: (c) examining the effect of the genetic modification on tumorformation, tumor growth, tumor cell proliferation, tumor cell survival,tumor stem cell survival, tumor stem cell proliferation, tumor cellcycle status, and tumor stem cell frequency.

In other embodiments, the present invention provides an in vivo methodfor proliferating a population of cancer cells, comprising: (a)introducing an enriched population of solid tumor stem cells into a hostanimal; wherein: (i) the tumor cells are derived from a solid tumor;(ii) the solid tumor stem cells are tumorigenic; (iii) the solid tumorstem cell population is enriched at least 2-fold relative tounfractionated tumor cells; (b) proliferating the solid tumor stem cellsin the host animal; and (c) purifying the proliferated solid tumor stemcells from the host animal. In certain embodiments, the cells areintroduced subcutaneously or into the mammary fat pad. In otherembodiments, the tumors grow in the mammal in greater than 75% of theintroductions. In further embodiments, the tumors grow in the mammal ingreater than 90% of the introductions. In other embodiments, thepopulation of cells has been enriched at least 50-fold. In someembodiments, the population of cells has been enriched at least 5-foldor at least 10-fold. In additional embodiments, the host animal is amouse. In certain embodiments, the mouse has immunosuppressed by amethod selected from the group consisting of administration of VP 16,radiation therapy and chemotherapy. In some embodiments, the methodsfurther comprise: (d) isolating an enriched population of solid tumorstem cells from the proliferated cells (e.g., by flow-cytometry).

In some embodiments, the present invention provides methods for growinga solid tumor stem cell from a solid tumor, comprising the steps of: (a)separating the cells of the solid tumor; (b) suspending the separatedtumor cells in suspension; and (c) introducing the suspended cell into ahost mammal, such that the cells in the introduced suspension forms atumor in the host mammal.

In certain embodiments, the present invention provides a tumor bank,comprising cells derived from a single tumor, wherein the tumor has beenproduced by the steps of: (a) introducing an enriched population ofsolid tumor stem cells into a host mammal; wherein: (i) the tumor cellsare derived from a solid tumor; (ii) the solid tumor stem cells aretumorigenic; and (iii) the solid tumor stem cell population is enrichedat least 2-fold relative to unfractionated tumor cells; (b)proliferating the introduced cells in the host mammal; and (c) isolatingthe proliferated cells from the host mammal.

In particular embodiments, the present invention provides a chimericanimal, comprising: (a) the animal; and (b) an enriched population ofsolid tumor stem cells, wherein: (i) the tumor cells are derived from asolid tumor; (ii) the solid tumor stem cells are tumorigenic; (iii) thesolid tumor stem cell population is enriched at least 2-fold relative tounfractionated tumor cells; and (iv) the cells of the enrichedpopulation have been introduced into the animal. In certain embodiments,the introduction is by injection.

In further embodiments, the present invention provides methods for an invivo method for modeling a tumor treatment regime, comprising the stepsof: (a) introducing an enriched population of solid tumor stem cellsinto an immunocompromised mouse under conditions that allow the solidtumor stem cells to proliferate to form a tumor; wherein: (i) the tumorcells are derived from a solid tumor; (ii) the solid tumor stem cellsare tumorigenic; and (iii) the solid tumor stem cell population isenriched at least 2-fold relative to unfractionated tumor cells; and (b)testing the effects of treatment regimens on the solid tumor cells inthe immunocompromised mouse by monitoring the effect of the treatmentregimen.

In certain embodiments, the present invention provides methods forreducing the size of a solid tumor, comprising the step of: contactingthe cells of the solid tumor with a therapeutically effective amount ofan agent directed against a Notch 4 polypeptide. In other embodiments,the therapeutically effective amount is an amount sufficient to causecell death of or inhibit the proliferation of solid tumor stem cells inthe solid tumor. In some embodiments, the agent is an antibody, peptideor small molecule directed against a Notch 4 polypeptide. In specificembodiments, the antibody, peptide or small molecule is directed againstthe extracellular domain of Notch 4.

In some embodiments, the present invention provides methods for reducingthe size of a solid tumor, comprising: contacting the cells of the solidtumor with a therapeutically effective amount of an agent that modulatesthe activity of a Notch 4 ligand. In other embodiments, the Notch 4ligand is selected from the group consisting of Delta 1, Delta 2,Delta-like ligand 4 (Dll4), Jagged 1 and Jagged 2. In furtherembodiments, the agent is a Notch ligand agonist. In other embodiments,the agent is a Notch ligand antagonist.

In particular embodiments, the present invention provides methods forreducing the size of a solid tumor, comprising: contacting the cells ofthe solid tumor with a therapeutically effective amount of an agent thatmodulates the activity of Maniac Fringe. In some embodiments, the agentis a Maniac Fringe agonist. In other embodiments, the agent is a ManiacFringe antagonist.

In certain embodiments, the present invention provides methods forkilling or inhibiting the proliferation of solid tumor stem cells,comprising the step of: contacting the cells of a solid tumor with anagent or combination of agents selectively targeted to the solid tumorstem cells of the solid tumor, wherein the agent or combination ofagents kills or inhibits the proliferation of solid tumor stem cells. Inother embodiments, the methods further comprise the step of: identifyingthe death of or the prevention of the growth of solid tumor stem cellsin the solid tumor following contact by the agent or combination ofagents. In some embodiments, the killing is by the activation of celldeath in the solid tumor stem cells. In particular embodiments, the celldeath is apoptosis. In additional embodiments, the agent or combinationof agents inhibits Notch-4 signaling. In particular embodiments, theagent is an antibody, peptide or small molecule directed against a Notch4 polypeptide. In some embodiments, the antibody, peptide or smallmolecule is directed against the extracellular domain of Notch 4. Infurther embodiments, the agent or combination of agents modulates theactivity of a Notch 4 ligand. In other embodiments, the Notch 4 ligandis selected from the group consisting of Delta 1, Delta 2, Delta-likeligand 4 (Dll4), Jagged 1 and Jagged 2. In further embodiments, theagent or combination of agents modulates the activity of Maniac Fringe.

In some embodiments, the present invention provides methods for reducingthe size of a solid tumor, comprising the step of: contacting the cellsof the solid tumor in vivo with an agent or combination of agentsselectively targeted to the solid tumor stem cells of the solid tumor,wherein the agent or combination of agents kills or inhibits theproliferation of solid tumor stem cells. In other embodiments, themethods further comprise the step of: identifying the death of or theprevention of the growth of solid tumor stem cells in the solid tumorfollowing contact by the agent or combination of agents. In additionalembodiments, the activation of cell death in the solid tumor stem cells.In other embodiments, the methods further comprise the step of:identifying the death of or the prevention of the growth of solid tumorstem cells in the solid tumor following contact by the agent orcombination of agents. In some embodiments, the killing is by theactivation of cell death in the solid tumor stem cells. In particularembodiments, the cell death is apoptosis. In additional embodiments, theagent or combination of agents inhibits Notch-4 signaling. In particularembodiments, the agent is an antibody, peptide or small moleculedirected against a Notch 4 polypeptide. In some embodiments, theantibody, peptide or small molecule is directed against theextracellular domain of Notch 4. In further embodiments, the agent orcombination of agents modulates the activity of a Notch 4 ligand. Inother embodiments, the Notch 4 ligand is selected from the groupconsisting of Delta 1, Delta 2, Delta-like ligand 4 (Dll4), Jagged 1 andJagged 2. In further embodiments, the agent or combination of agentsmodulates the activity of Maniac Fringe.

In further embodiments, the present invention provides methods forselectively targeting a solid tumor stem cell, comprising the steps of:(a) identifying a marker present on a solid tumor stem cell; (b)obtaining a biomolecule or set of biomolecules that selectively binds tothe marker present on the solid tumor stem cell. In certain embodiments,the biomolecule genetically modifies the targeted solid tumor stem cell.In some embodiments, the genetic modification results in solid tumorstem cell death. In other embodiments, the biomolecule or set ofbiomolecules comprises a bi-specific conjugate. In particularembodiments, the biomolecule or set of biomolecules comprises anadenoviral vector. In other embodiments, the adenoviral vector isselectively targeted to a solid tumor stem cell.

In some embodiments, the present invention provides a biomolecule or setof biomolecules that is selectively targeted to solid tumor stem cell.In other embodiments, the biomolecule genetically modifies the targetedsolid tumor stem cell. In further embodiments, the genetic modificationresults in solid tumor stem cell death. In particular embodiments, thebiomolecule or set of biomolecules comprises a bi-specific conjugate. Insome embodiments, the biomolecule or set of biomolecules comprises anadenoviral vector. In other embodiments, the adenoviral vector isselectively targeted to a solid tumor stem cell.

In certain embodiments, the present invention provides methods forforming a tumor in an animal, comprising: introducing a cell dose ofpurified solid tumor stem cells into the animal, wherein: (a) the solidtumor stem cell is derived from a solid tumor; (b) the solid tumor stemcell population is enriched at least 2-fold relative to unfractionatedtumor cells. In certain embodiments, the number of cells in the celldose is between about 100 cells and about 5×10⁵ cells. In otherembodiments, the number of cells in the cell dose is about between about100 cells and 500 cells. In further embodiments, the number of cells inthe cell dose is between about 100 cells and 200 cells. In someembodiments, the number of cells in the cell dose is about 100 cells.

In particular embodiments, a culture medium is provided in which culturemedium the solid tumor stem cell is situated. In other embodiments, theculture medium comprises a Notch ligand. In certain embodiments, thesolid tumor stem cell is affixed to a substrate. In other embodiments,the solid tumor stem cell has been treated to reduce proliferation. Infurther embodiments, the solid tumor stem cell has been treated toincrease proliferation.

DESCRIPTION OF THE FIGURES

FIG. 1 shows two models of solid tumor heterogeneity. In the classicmodel (FIG. 1A), mutations or environmental differences cause tumorcells to adopt a variety of different phenotypes. Environmentallydetermined differences in phenotype, represented by white, green, andred cells, may be reversible while mutationally determined changes inphenotype, represented by purple cells, may not be reversible. Manycells with a variety of different phenotypes are thought to have thepotential to proliferate extensively and form new tumors. The tumor stemcell model (FIG. 1B) is distinguished by having only a minor populationof tumor cells that are tumorigenic (yellow cells). These tumor stemcells are characterized by indefinite proliferative potential, theability to form new tumors, and the ability to give rise toheterogeneous non-tumorigenic cancer cells that typically form the bulkof a tumor.

FIG. 2 is a set of FACS plots of breast cancer tumor cells. Mice wereimplanted with primary breast cancer tumor cells removed from two humanpatients. Resultant tumors were removed from the mouse and single cellsuspensions were made. Cells were stained with anti-CD44-PE,anti-520C9-APC, anti-mouse H2K-FITC (which stains infiltrating mousecells) and Propidium Iodide (PI, which stains dead cells). FIGS. 2A and2B show FACS plots of tumor cells obtained from mice implanted withprimary breast cancer tumor cells removed from human Patient 1 and humanPatient 2, respectively. Live, human CD44+ and human CD44− cells wereisolated and used for in vitro and in vivo studies.

FIG. 3 is a set of FACS plots showing the expression of CD24 bymalignant breast cells. Cells were isolated and stained as described inFIG. 2. Mouse cells and dead cells were gated out of the analysis. TheFACS plots of cells from three breast cancer tumors are shown (FIGS. 3A,3B, and 3C, respectively). Note that cells from all three tumors have asimilar phenotype.

FIG. 4 is a set of FACS plots showing an analysis of tumors arising fromthe CD24⁻ cell population from human breast cancers. According to thesolid tumor stem cell model, the CD24⁻ cells give rise to tumors thatcontain both CD24⁺ and CD24⁻ cells. Accordingly, secondary transplantswere performed using B38.1⁺CD24⁻ cells (FIG. 4A). The resultant tumorswere removed and the cells were re-analyzed with respect to B38.1 andCD24 expression. As predicted by the stem cell model, cells obtainedfrom a tumor arising from transplanted B38.1⁺CD24⁻ cells wereheterogeneous with respect to expression of both B38.1 and CD24 (FIG.4B). The marker expression pattern of the cells isolated from the tumorinitiated by the B38.1⁺CD24⁻ cells was similar to that of the originaltumor (FIG. 4).

FIG. 5 is a FACS plot showing an analysis of Notch 4 expression. Cellswere isolated from a mouse xenograft tumor (see, below) and stained withantibodies. Malignant cells were analyzed for expression of B38.1 andNotch 4. Mouse cells and dead cells were gated out of the analysis.

FIG. 6 shows the fractionation of breast cancer cells based upon CD44expression. Tumor T1 cells (FIG. 6A, FIG. 6C, and FIG. 6E) and Tumor T2cells (FIG. 6B, FIG. 6D, and FIG. 6F) were stained with anti-CD44-PE,anti-mouse H2K-FITC and the viability dye 7AAD. Flow cytometry was usedto isolate live, human (H2K−) cells that were either CD44⁺ (FIG. 6C,FIG. 6D) or CD44⁻ (FIG. 6E, FIG. 6F). Dead cells (7AAD⁺) were eliminatedfrom all analyses. FIG. 6A and FIG. 6B are dot plots of theunfractionated T1 and T2 cells showing CD44 and H2K expression asindicated. Plots showing the isolated CD44⁺ (FIG. 6C, FIG. 6D) and CD44⁻(FIG. 6E, FIG. 6F) populations depict reanalyses of cells that had beenisolated by flow-cytometry. These cells were injected into the mammaryfat pads of mice to examine their tumorigenicity. TABLES 1 and 3 showthat the CD44⁺ cells but not the CD44⁻ cells were tumorigenic.

FIG. 7 shows the isolation of tumorigenic cells. Flow cytometry was usedto isolate subpopulations of Tumor T1 (FIG. 7A, FIG. 7D, and FIG. 7G),Tumor T2 (FIG. 7B, FIG. 7E, and FIG. 7F) or Tumor T5 cells (FIG. 7C,FIG. 7F, and FIG. 7I) that were tested for tumorigenicity in NOD/SCIDmice. T1 and T2 cells had been passaged once in NOD/SCID mice while T5cells were obtained from material that had been frozen immediately afterremoval from a patient. Cells were stained with anti-B38.1-APC,anti-CD44-PE, anti-CD24-FITC, anti-LINEAGE-Cytochrome,anti-mouse-H2K-Cytochrome (T1 and T2 cells only), and 7AAD. Dead cells(7AAD⁺), mouse cells (H2K⁺) and LINEAGE⁺ cells were eliminated from allanalyses. Each dot plot depicts the CD24 and CD44 staining patterns oflive human B38.1⁺LINEAGE⁻ cells. FIG. 7A, FIG. 7B, and FIG. 7C showunfractionated tumor cells. B38.1⁺CD44⁺LINEAGE⁻ cells that were eitherCD24^(−/lo) (FIG. 7G, FIG. 7H, FIG. 7I) or CD24⁺ (FIG. 7D, FIG. 7E, FIG.7F) were isolated from these tumor cells by flow-cytometry. FIGS. 7D-7Idepict reanalyses of these sorted populations, which were subsequentlyinjected into the mammary fat pads of NOD/SCID mice to testtumorigenicity. FIG. 7J shows a representative tumor in a mouse at theB38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ injection site, but not at theB38.1⁺CD44⁺CD24⁺LINEAGE⁻ injection site. Histology performed on tissuefrom the CD24⁺ (FIG. 7K, 20× objective magnification) and CD24^(−/lo)(FIG. 7L, 40× objective magnification) injection sites exhibited normalmouse tissue and malignant cells respectively.

FIG. 8 shows the enrichment of tumorigenic cells based upon ESAexpression. Flow cytometry was used to isolate subpopulations of TumorT1 cells that were tested for tumorigenicity in NOD/SCID mice. T1 cellshad been passaged once in NOD/SCID mice. Cells were stained withanti-B38.1-APC, anti-CD24-PE, anti-ESA-FITC, anti-LINEAGE-Cytochrome,anti-mouse-H2K-Cytochrome (T1), and 7AAD. Dead cells (7AAD⁺), mousecells (H2K⁺) and LINEAGE⁺ cells were eliminated from the analysis. Thedot plot in FIG. 8A depicts the CD24 and ESA staining pattern of livehuman B38.1⁺LINEAGE⁻ cells. The tumorigenic population is boxed andmarked with an arrow. In FIG. 8B, the ESA⁺B38.1⁺CD24^(−/lo)LINEAGE⁻cells (left panel) and the remaining LINEAGE⁻ H2K⁻ cells (right panel)were collected using flow cytometry.

FIG. 9 is the results of an in vitro clonogenic assay. Flow cytometrywas used to isolate tumorigenic cell or the rest of the non-tumorigenicneoplastic (non-tumorigenic cells) as described. The cells were placedin tissue culture medium containing soluble Delta for the indicatednumber of days. The tumorigenic and non-tumorigenic xenograft Tumor 1(T1) (FIG. 9A), Tumor 4 (T4) (FIG. 9B) or primary patient (FIG. 9C)cells are shown at the indicated time after being placed in tissueculture. T4 cells were stained with Papanicolaou stain and examinedunder light microscopy (100× objective). Note that both thenon-tumorigenic (FIG. 9D) and tumorigenic (FIG. 9E) populations consistof neoplastic cells with large nuclei and prominent nucleoli. Note thatthe number of cells that attached to the tissue culture plate is similarin both populations, but that the tumorigenic population always gaverise to colonies. Non-tumorigenic populations do not give rise toestablished colonies (or only for brief periods, about 2-6 days).

FIG. 10 is a set of dot plots showing the phenotypic diversity in tumorsarising from B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells. The dot plots depictthe CD24 and CD44 staining patterns of live human LINEAGE⁻ cells fromTumor T1 (FIG. 10A-FIG. 10C) or Tumor T2 (FIG. 10D-FIG. 10F). FIG. 10Aand FIG. 10D show unfractionated T1 or T2 cells obtained from tumorsthat had been passaged once in NOD/SCID mice.B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells from T1 (FIG. 10B) or T2 (FIG. 10E)were isolated as described in FIG. 2, above. TheB38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ populations (reanalyzed in FIG. 10B andFIG. 10E) were injected into the mammary fat pads of NOD/SCID mice. FIG.10C and FIG. 10F depict analyses of the tumors that arose from theseB38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells. Note that in both cases, theB38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells formed tumors that contained aphenotypically diverse population of cells similar to that observed inthe original tumor.

FIG. 11 shows the expression of HER2/neu and EGF-R. Flow cytometry wasused to isolate subpopulations of Tumor T1 cells that had been passagedonce in NOD/SCID mice. Cells were stained with, in FIG. 11A,anti-EGF-R-PE, anti-B38.1-APC, anti-CD24-FITC, anti-LINEAGE-Cytochrome,anti-mouse-H2K-Cytochrome, and 7AAD or, in FIG. 11B anti-HER2/neu-FITC,anti-B38.1-APC, anti-CD24-PE, anti-LINEAGE-Cytochrome,anti-mouse-H2K-Cytochrome, and 7AAD. Dead cells (7AAD⁺), mouse cells(H2K⁺) and LINEAGE⁺ cells were eliminated from all analyses. Thehistogram in FIG. 11A depicts the EGF-R expression of the unstainedcells (dotted line), B38.1⁺CD24⁻LINEAGE⁻ tumorigenic population (shaded)and the B38⁺CD24⁺LINEAGE⁻ non-tumorigenic (solid line) population. Thehistogram in FIG. 11B shows HER2/neu expression of the unstained cells(dotted line), B38.1⁺CD24⁻LINEAGE⁻ tumorigenic population (shaded) andthe B38⁺CD24⁺LINEAGE⁻ non-tumorigenic (solid line) population. RT-PCRwas performed using nested primers to detect EGF-R (FIG. 11C and FIG.11D) or to detect HER2/neu (FIG. 11E). One cell per sample in panelsFIG. 11D and FIG. 11E, or ten cells per sample in panel FIG. 11C, wereanalyzed. EGF-R is expressed at lower levels in tumorigenic cells thanin non-tumorigenic cells at both the protein (FIG. 11A) and mRNA levels(FIG. 11C, FIG. 11D).

FIG. 12 is a photomicrograph of breast cancer cells placed in tissueculture after exposure to an anti-Notch 4 antibody. Cells were incubatedon ice for one hour in HBSS with or without soluble Delta but noanti-Notch 4 antibody, with anti-Notch 4 antibody, or with anti-Notch 4antibody that had been preincubated with the peptide used to generatethe antibody. The number of colonies that formed in the triplicateexperiments is shown. Soluble Delta was added to the culture. Fc-controlmedium without soluble Delta was added to the culture. Symbols: Ab=theanti-Notch 4 antibody; Block=the peptide used to generate the anti-Notch4 antibody.

FIG. 13 is a schematic diagram of B38.1⁺ cells within a tumor. Thebreast cancer stem cells from multiple patients are B38.1⁺. Tosuccessfully treat a cancer with a gene therapy approach, these cellscan be targeted with a vector.

FIG. 14 is a description of the method for obtaining the bi-specificconjugate and the chemical modifications introduced in the antibodies.

FIG. 15 is a strategy for re-targeting Adenovirus. The LaZ virus caninfect most of the cells from a tumor. After the LaZ virus is incubatedonly with the anti-fiber antibody, the LaZ virus loses ability to infectall of the cells. After the LaZ virus is incubated with the bi-specificconjugate, the B38.1 moiety of the molecule allows the attachment of thevirus to the B38.1⁺ cells, so only these cells are infected.

FIG. 16 shows the targeting of breast cancer stem cells with thebi-specific antibody. Different cell lines were infected with AdLaZ,which is an E1-deleted Adenovirus that expresses the β-galactosidasegene (gray columns, control for virus infection). In some cases, thevirus was incubated with the anti-fiber antibody for 30 min beforeinfection (yellow columns). In other cases, the virus was incubated withthe bi-specific conjugate (green columns). After 24 hr of infection, themonolayers were fixed and incubated with X-Gal-contained buffer. Theinfected cells are blue, and the graphic shows the percentage of bluecells obtained, relative to the control infection (i.e., the reductionor increase in infectivity of the virus after incubation with thedifferent antibodies).

FIG. 17 shows that the bispecific antibody can target an adenovirusvector to breast cancer stem cells. The columns represent the absolutenumber of infected cells per field. Gray: The indicated cells infectedwith the control adenovirus. Yellow: the indicated cells were infectedwith the adenovirus that had been incubated with the anti-fiberantibody. Green: The indicated cells were incubated with the bi-specificconjugate antibody. Red: Cells were infected with the virus that hadbeen incubated with the bi-specific conjugate antibody, but the cellswere pre-treated with an excess of B38.1 antibody.

FIG. 18 is a photograph of some of the cell monolayers after X-Galstaining. The infected cells appear like dark dots in this black andwhite picture (the β-galactosidase gene of the LaZ virus has a nuclearlocalization signal. The staining is in the nuclei of the cells.

FIG. 19 is an analysis if different populations of cells in a breastcancer. ESA⁺CD44⁺CD24⁻/loLINEAGE⁻ cells breast cancer stem cells (FIG.19A) and ESA⁺CD44⁺CD24⁺LINEAGE-non-tumorigenic cells (FIG. 19B) wereobtained as described in FIG. 8. The cells were stained with Hoechst33342 as described by Eaves and colleagues (Glimm H et al., Blood.96(13): 4185-93 (2000)). The histogram for the breast cancer stem cellsis shaded. Note that the breast cancer stem cells and thenon-tumorigenic cells are distributed in all phases of the cells cycle.

FIG. 20 is a further analysis of different populations of cells in abreast cancer. CD44⁺CD24^(−/lo)LINEAGE⁻ cells breast cancer stem cellsand non-tumorigenic CD44⁺CD24⁺LINEAGE⁻ non-tumorigenic cells wereobtained as described in FIG. 7. The cells were stained with Rhodamine123 as described by Spangrude et al., Blood 85(4):1006-16, 1995). Thehistogram for the breast cancer stem cells is shaded. Note that thebreast cancer stem cells tend to stain less intensely with Rhodamine123.

FIG. 21 is an analysis of ascites fluid for ovarian cancer stem cells.Cells were stained with anti-B38.1-APC, anti-CD44-PE, anti-CD24-FITC,anti-Lineage-Cytochrome and 7AAD. Dead cells (7AAD⁺), and LINEAGE⁺ cellswere eliminated from the analyses. Note that there is a distinctCD44⁺CD24^(−/lo)LINEAGE⁻ population of cells that resembles the breastcancer stem cells.

FIG. 22 is an analysis of sarcoma cells for solid tumor stem cells. P1sarcoma cells growing in the xenograft model were stained withanti-B38.1-APC, anti-CD44-PE, anti-CD24-FITC, anti-LINEAGE-Cytochrome,anti-H2K-Cytochrome and 7AAD. Dead cells (7AAD⁺), LINEAGE⁺ cells andmouse cells were eliminated from the analyses. Note that the lineagecocktail in this analysis did not include CDIO, CD31 or CD140b. Alsonote that there is a distinct CD44⁺CD24^(−/lo)LINEAGE⁻ population ofcells.

FIG. 23: The Majority of CD44+ Cells have Elevated ALDH Activity. A)CD44/Aldefluor™ flow cytometry profile of mouse lineage-negative (mLin−;H2 Kd and murine CD45), ESA+ xenogeneic UM-C4 and UM-C6 colon tumorcells in the presence or absence of the ALDH1 inhibitor, DEAB. Tumorgrowth curves are shown for the denoted FACS purified populations fromB) UM-C4 and C) UM-C6 tumors injected at a dose of 500 cellssubcutaneously. Means+SEM are plotted and reflect only those mice withpalpable tumors.

FIG. 24: Relative ALDH Gene Family Expression. A) Absolute expressionlevels of ALDH family member mRNA as determined by Affymetrix microarryanalysis is shown. B) Inset shows the relative expression of severalALDH genes in TG versus NTG cells from different colon tumor xenografts.C) Inset shows relative expression of ALDH1A1 as validated by Taqmanquantitative RT-PCR using FACS-purified TG versus NTG cells. Resultswere normalized versus an internal control (i.e. GUSB) prior to relativeexpression analysis.

FIG. 25: Tumorigenic UM-C4 Cells Preferentially Survive CPAChemotherapy. A) Twice weekly administration of 25 mg/kg CPA resulted indelayed UM-C4 tumor growth versus control, vehicle-treated mice.Phenotypic analysis of residual tumors at day fifteen demonstrated B)higher concentrations of ESA+CD44+ cells and C) an increased percentageof cells with ALDH activity. D) Limiting dilution analysis ofunfractionated UM-C4 tumor cells demonstrated a significant increase intumorigenic cell frequency. E) Though ESA+CD44+ cells were equallytumorigenic, tumors were more aggressive when obtained from CPA comparedto control tumors.

FIG. 26: Tumors Treated with Anti-Integrin Beta 1 Antibodies ShowDecreased Numbers of Tumorgenic Cells. Immunocompromised mice (n=10)were injected with decreasing dosages of tumor cells treated with eithercontrol (C) or AIIB2 antibodies (T). Tumor volume (mm2) was graphedcompared to the number of human tumor cells injected: 1000, 333, 111,and 37. A) Results of tumor take rates on day 28 (D28). The number ofanimals with detectable tumors over the ten injected animals for eachtumor cell dose is recorded below the graph of tumor volume with controltreated tumor cells on the left (black) and AIIB2 treated tumor cells onthe right (red) for each cell dose. B) Results of tumor take rates onday 34 (D34). The number of animals with detectable tumors over the teninjected animals for each tumor cell dose is recorded below the graph oftumor volume with control treated tumor cells on the left (black) andAIIB2 treated tumor cells on the right (red) for each cell dose. C)Results of tumor take rates on day 42 (D42). The number of animals withdetectable tumors over the ten injected animals for each tumor cell doseis recorded below the graph of tumor volume with control treated tumorcells on the left (black) and AIIB2 treated tumor cells on the right(red) for each cell dose. D) The stem cell frequency on day 28 wascalculated. The proportion of stem cells (y-axis) from control treated(open bar, left) compared to AIIB2 treated (filled bar, right) isplotted and the 95% confidency interval show (*).

GENERAL DESCRIPTION OF THE INVENTION

A small percentage of cells within an established solid tumor have theproperties of stem cells. These solid tumor stem cells give rise both tomore tumor stem cells and to the majority of cells in the tumor thathave lost the capacity for extensive proliferation and the ability togive rise to new tumors. Thus, solid tumor heterogeneity reflects thepresence of tumor cell progeny arising from a solid tumor stem cell. Thepresent invention provides a xenograft model which can be used toestablish tumors from primary tumors via injection of tumors in themammary gland of severely immunodeficient mice. These xenograft assayhave allowed biological and molecular assays to characterize clonogenicsolid tumor stem cells. The present invention also provides evidencethat strongly implicates the Notch pathway, especially Notch 4, asplaying a central pathway in carcinogenesis.

The invention is based upon the discovery that a small percentage ofcells within an established solid tumor have the properties of “stemcells”. These solid tumor “stem” cells give rise both to more solidtumor stem cells and to the majority of cells in the tumor, cancer cellsthat have lost the capacity for extensive proliferation and the abilityto give rise to new tumors. Thus, solid tumor cell heterogeneityreflects the presence of a variety of tumor cell types that arise from asolid tumor stem cell.

The previous failure of cancer therapies to significantly improveoutcome has been due in part to the failure of these therapies to targetthe solid tumor stem cells within a solid tumor that have the capacityfor extensive proliferation and the ability to give rise to all othersolid tumor cell types. This invention provides a way that anti-cancertherapies can be directed, both generally and now specifically directed,against the solid tumor stem cells. The directed anti-cancer therapiesof the invention thus result in much more effective and durabletherapeutic responses.

By the methods of the invention, one can characterize the phenotypicallyheterogeneous populations of cells within a solid tumor. Populations ofcells obtained from the solid tumor are isolated and structurallycharacterized using Fluorescence Activated Cell Sorting (FACS). Inparticular, one can identify, isolate, and characterize a phenotypicallydistinct cell population within a tumor having the stem cell propertiesof extensive proliferation and the ability to give rise to all othertumor cell types. Solid tumor stem cells are the truly tumorigenic cellsthat are capable of re-establishing a tumor following treatment.

The invention provides in vivo and in vitro assays of solid tumor stemcell function and cell function by the various populations of cellsisolated from a solid tumor. The invention provides methods for usingthe various populations of cells isolated from a solid tumor (such as apopulation of cells enriched for solid tumor stem cells) to identifyfactors influencing solid tumor stem cell proliferation, to analyzepopulations of cells isolated from solid tumors for gene expressionpatterns or protein expression patterns, to identify new anti-cancerdrug targets, to predict the sensitivity of tumors from individualpatients to existing anti-cancer treatment regimens, to modelanti-cancer treatment, to test new therapeutic compounds, to identifyand test new diagnostic markers, to treat tumors, to produce geneticallymodified solid tumor stem cells, and to prepare cDNA libraries andmicroarrays of polynucleotides and polypeptides from solid tumor stemcells.

The invention provides a method for consistently growing solid tumorcells in vivo. The invention also provides a method to grow solid tumorcells that are in single cell suspension or in small aggregates.Moreover, the invention provides a chimeric animal (a xenograft model)in which tumors can be established from solid tumor primary cells and inwhich the tumors derived from these solid tumor cells can be tested.Furthermore, the invention provides tumor banks (large enough to performsubstantial numbers of bioassays) derived from single solid tumor stemcells.

In its several aspects, the invention usefully provides methods forscreening for anti-cancer agents; for the testing of anti-cancertherapies; for the development of drugs targeting novel pathways; forthe identification of new anti-cancer therapeutic targets; theidentification and diagnosis of malignant cells in pathology specimens;for the testing and assaying of solid tumor stem cell drug sensitivity;for the measurement of specific factors that predict drug sensitivity;and for the screening of patients (e.g., as an adjunct for mammography).The invention can be used as a model to test patients' tumor sensitivityto known therapies; as a model for identification of new therapeutictargets for cancer treatment; as a system to establish a tumor bank fortesting new therapeutic agents for treatment of cancer; and as a systemto identify the tumorigenic cancer cells. Also, the invention providessynergy between the methods of the invention and breast cancer genomicdatabases, for an improved anti-cancer drug discovery.

DETAILED DESCRIPTION OF THE INVENTION

Stem cells and solid tumor heterogeneity models. Solid tumors arecomposed of heterogeneous cell populations. For example, breast cancersare a mixture of cancer cells and normal cells, including mesenchymal(stromal) cells, inflammatory cells, and endothelial cells. Classicmodels hold that phenotypically distinct cancer cell populations allhave the capacity to proliferate and give rise to a new tumor (FIG. 1A).In the classical model, tumor cell heterogeneity results fromenvironmental factors as well as ongoing mutation within cancer cellsresulting in diverse populations of tumorigenic cells and allpopulations of cells would have similar tumorigenic potential. Pandis etal., Genes, Chromosomes & Cancer 12:122-129 (1998); Kuukasjrvi et al.,Cancer Res. 57: 1597-1604 (1997); Bonsing et al., Cancer 71: 382-391(1993); Bonsing et al., Genes Chromosomes & Cancer 82: 173-183 (2000);Beerman H et al., Cytometry. 12(2): 147-54 (1991); Aubele M & Werner M,Analyt. Cell. Path. 19: 53 (1999); Shen L et al., Cancer Res. 60: 3884(2000).).

This invention is based upon an alternative model of solid tumor cellheterogeneity, in which a solid tumor results from a “solid tumor stemcell” (or “cancer stem cell” from a solid tumor) and the subsequentchaotic development of the solid tumor stem cell. In this stem cellmodel (FIG. 1B), solid tumors contain a distinct, limited (or possiblyrare) subset of cells that share the properties of normal “stem cells”,in that they proliferate extensively or indefinitely and that theyefficiently give rise to additional solid tumor stem cells. Within anestablished solid tumor, most cells have lost the ability to proliferateextensively and form new tumors, but solid tumor stem cells proliferateextensively and give rise to additional solid tumor stem cells as wellas to other tumor cells that lack tumorigenic potential. It is thissolid tumor stem cell population that proliferates and ultimately provesfatal.

To distinguish between these models, the deficiencies of the previousclonogenic assays (see, below) must be overcome. To prove the existenceof a consistent stem cell population rather that a constant lowprobability of tumorigenicity in any cell type, one must be able topurify the stem cells and show that they are highly enriched fortumorigenicity, while the remainder of the neoplastic cells are depletedof such activity. The invention provides this ability.

The ability to isolate and analyze cell populations within a solidtumor, based upon structural features of the solid tumor stem cells,described herein, allows one skilled in the art of oncology or stem cellbiology to distinguish between the two models shown in FIG. 1. By thisinvention, solid tumor stem cells and cell populations from solid tumorshave been isolated and analyzed. Moreover, these solid tumor stem cellshave very high or unlimited proliferative potential, and thus representthe truly tumorigenic population. According to the solid tumor stem cellmodel and the results provided below (see, EXAMPLES), these tumorigeniccells are the clonogenic cells of solid tumors.

During normal animal development, cells of most or all tissues arederived from normal precursors, called stem cells (Morrison et al., Cell88(3): 287-98 (1997); Morrison et al., Curr. Opin. Immunol. 9(2): 216-21(1997); Morrison et al., Annu. Rev. Cell. Dev. Biol. 11:35-71 (1995)).The term “stem cell” is known in the art to mean (1) that the cell is acell capable of generating one or more kinds of progeny with reducedproliferative or developmental potential; (2) that the cell hasextensive proliferative capacity; and (3) that the cell is capable ofself-renewal or self-maintenance (see, Pollen et al., Development 110:1001 (1990); U.S. Pat. Nos. 5,750,376, 5,851,832, 5,753,506, 5,589,376,5,824,489, 5,654,183, 5,693,482, 5,672,499, and 5,849,553, allincorporated by reference). In adult animals, some cells (includingcells of the blood, gut, breast ductal system, and skin) are constantlyreplenished from a small population of stem cells in each tissue. Thus,the maintenance of tissues (whether during normal life or in response toinjury and disease) depends upon the replenishing of the tissues fromprecursor cells in response to specific developmental signals.

The best-known example of adult cell renewal by the differentiation ofstem cells is the hematopoietic system (see, U.S. Pat. Nos. 5,061,620,5,087,570, 5,643,741, 5,821,108, 5,914,108, each incorporated byreference). Developmentally immature precursors (hematopoietic stem andprogenitor cells) respond to molecular signals to gradually form thevaried blood and lymphoid cell types. Stem cells are also found in othertissues, including epithelial tissues (see, Slack, Science 287: 1431(2000)) and mesenchymal tissues. (see, U.S. Pat. No. 5,942,225;incorporated by reference). In normal breast development, a normal stemcell gives rise to differentiated progeny to form a normal ductalsystem. Kordon & Smith, Development 125: 1921-1930 (1998); see also,U.S. Pat. Nos. 5,814,511 and 5,650,317.

By this invention, the principles of normal stem cell biology have beenapplied to isolate and characterize solid tumor stem cells. Examples ofsolid tumors from which solid tumor stem cells can be isolated orenriched for according to the invention include sarcomas and carcinomassuch as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. The invention is applicable to sarcomas (see, FIG. 22)and epithelial cancers, such as ovarian cancers (see, FIG. 21) andbreast cancers (see, EXAMPLES).

Solid tumor stem cells are defined structurally and functionally asdescribed herein; using the methods and assays similar to thosedescribed below. Because tumor cells are known to evolve phenotypicallyand functionally over time as additional genetic mutations occur, thesolid tumor stem cells may change phenotypically and functionally overtime in an individual patient. Nevertheless, one can use the method ofthe invention, employing the markers disclosed herein, which areconsistently useful in the isolation or identification of solid tumorstem cells in a majority of patients.

Also, solid tumor stem cells undergo “self-renewal” and“differentiation” in a chaotic development to form a tumor, give rise toabnormal cell types, and may change over time as additional mutationsoccur. The functional features of a solid tumor stem cell are that theyare tumorigenic, they give rise to additional tumorigenic cells(“self-renew”), and they can give rise to non-tumorigenic tumor cells(“differentiation”).

The developmental origin of solid tumor stem cells can vary betweendifferent types of solid tumor cancers. Solid tumor stem cells may ariseeither as a result of genetic damage that deregulates the proliferationand differentiation of normal stem cells (Lapidot et al., Nature367(6464): 645-8 (1994)) or by the dysregulated proliferation of anormal restricted progenitor or a normal differentiated cell type.Typically, solid tumors are visualized and initially identifiedaccording to their locations, not by their developmental origin.

By contrast, a non-tumorigenic cell from a solid tumor is a cell from apopulation that fails to form a palpable tumor upon transplantation intoan immunocompromised mouse, wherein if the same number ofunfractionated, dissociated tumor cells were transplanted under the samecircumstances, the solid tumor stem cells would form a palpable tumor inthe same period of time. Thus non-tumorigenic cells are depleted fortumor forming activity in an animal model.

A “palpable tumor” is known to those in the medical arts as a tumor thatis capable of being handled, touched, or felt.

Because the tumorigenic changes are intrinsic to solid tumor stem cells,even after they have been removed from their normal environment withinthe tumor, the invention provides several novel uses:

(1) by identifying the genes and proteins expressed by solid tumor stemcells it is possible to identify proteins whose function is necessaryfor tumorigenesis and which represent novel drug targets;

(2) by purifying solid tumor stem cells based on phenotypic markers itis possible to study their gene expression patterns and functions muchmore directly and efficiently;

(3) by developing in vitro and in vivo assays of solid tumor stem cellfunction it is possible to more effectively test the effects ofpotential therapeutic compounds;

(4) by identifying markers of solid tumor stem cells it is possible tomore effectively diagnose the presence of malignant cells (even thosethat do not depend on rare environmental characteristics for theirability to make tumors); and

(5) by isolating solid tumor stem cells from individual patients andtransplanting them into in vitro and in vivo functional assays it ispossible to test the effectiveness of different drug regimens againstthem. Thus, it is possible to predict drug sensitivity and drugresistance.

The solid tumor stem cells of the model of the invention differs fromthe “cancer stem line” provided by U.S. Pat. No. 6,004,528. In thatpatent, the “cancer stem line” is defined as a slow growing progenitorcell type that itself has few mutations but which undergoes symmetricrather than asymmetric cell divisions as a result of tumorigenic changesthat occur in the cell's environment. This “cancer stem line” hypothesisthus proposes that highly mutated, rapidly proliferating tumor cellsarise largely as a result of an abnormal environment, which causesrelatively normal stem cells to accumulate and then undergo mutationsthat cause them to become tumor cells. U.S. Pat. No. 6,004,528 proposesthat such a model can be used to enhance the diagnosis of cancer. Thesolid tumor stem cell model is fundamentally different than the “cancerstem line” model and as a result exhibits utilities not offered by the“cancer stem line” model. First, solid tumor stem cells are not“mutationally spared”. The “mutationally spared cancer stem line”described by U.S. Pat. No. 6,004,528 may be considered a pre-cancerouslesion, while the solid tumor stem cells described of this invention arecancer cells that themselves contain the mutations that are responsiblefor tumorigenesis. That is, the solid tumor stem cells (“cancer stemcells”) of the invention would be included among the highly mutatedcells that are distinguished from the “cancer stem line” in U.S. Pat.No. 6,004,528. Second, the genetic mutations that lead to cancer arelargely intrinsic within the solid tumor stem cells rather than beingenvironmental. The solid tumor stem cell model predicts that isolatedsolid tumor stem cells can give rise to additional tumors upontransplantation (thus explaining metastasis) while the “cancer stemline” model would predict that transplanted “cancer stem line” cellswould not be able to give rise to a new tumor, since it was theirabnormal environment that was tumorigenic. Indeed, the ability totransplant dissociated, and phenotypically isolated human solid tumorstem cells to mice (into an environment that is very different from thenormal tumor environment), where they still form new tumors,distinguishes the present invention from the “cancer stem line” model.Third, solid tumor stem cells likely divide both symmetrically andasymmetrically, such that symmetric cell division is not an obligateproperty. Fourth, solid tumor stem cells may divide rapidly or slowly,depending on many variables, such that a slow proliferation rate is nota defining characteristic.

As described above, solid tumor stem cells can be operationallycharacterized by cell surface markers. These cell surface markers can berecognized by reagents that specifically bind to the cell surfacemarkers. For example, proteins, carbohydrates, or lipids on the surfacesof solid tumor stem cells can be immunologically recognized byantibodies specific for the particular protein or carbohydrate (forconstruction and use of antibodies to markers, see, Harlow, UsingAntibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 1999); see also, EXAMPLES). The set of markers present onthe cell surfaces of solid tumor stem cells (the “cancer stem cells” ofthe invention) and absent from the cell surfaces of these cells ischaracteristic for solid tumor stem cells. Therefore, solid tumor stemcells can be selected by positive and negative selection of cell surfacemarkers. A reagent that binds to a solid tumor stem cell is a “positivemarker” (i.e., a marker present on the cell surfaces of solid tumor stemcells) that can be used for the positive selection of solid tumor stemcells. A reagent that binds to a solid tumor stem cell “negative marker”(i.e., a marker not present on the cell surfaces of solid tumor stemcells but present on the surfaces of other cells obtained from solidtumors) can be used for the elimination of those solid tumor cells inthe population that are not solid tumor stem cells (i.e., for theelimination of cells that are not solid tumor stem cells).

In one embodiment, the discrimination between cells based upon thedetected expression of cell surface markers is by comparing the detectedexpression of the cell surface marker as compared with the meanexpression by a control population of cells. For example, the expressionof a marker on a solid tumor stem cell can be compared to the meanexpression of the marker by the other cells derived from the same tumoras the solid tumor stem cell. Other methods of discriminating amongcells by marker expression include methods of gating cells by flowcytometry based upon marker expression (see, Givan A, Flow CytometryFirst Principles, (Wiley-Liss, New York, 1992); Owens M A & Loken M R.,Flow Cytometry: Principles for Clinical Laboratory Practice,(Wiley-Liss, New York, 1995)).

Solid tumor stem cell positive markers may also be present on cellsother than solid tumor stem cells. Solid tumor stem cell negativemarkers may also be absent from cells other than solid tumor stem cells.While it is rare to identify a single marker that identifies a stemcell, it has often been possible to identify combinations of positiveand negative markers that uniquely identify stem cells and allow theirsubstantial enrichment in other contexts. Morrison et al., Cell 96(5):737-49 (1999); Morrison et al., Proc. Natl. Acad. Sci. USA 92(22):10302-6 (1995); Morrison & Weissman, Immunity 1(8): 661-73 (1994).

A “combination of reagents” is at least two reagents that bind to cellsurface markers either present (positive marker) or not present(negative marker) on the surfaces of solid tumor stem cells, or to acombination of positive and negative markers (see, EXAMPLES 7 and 8,TABLE 6). The use of a combination of antibodies specific for solidtumor stem cell surface markers results in the method of the inventionbeing useful for the isolation or enrichment of solid tumor stem cellsfrom a variety of solid tumors, including sarcomas, ovarian cancers, andbreast tumors. Guidance to the use of a combination of reagents can befound in PCT patent application WO 01/052143 (Morrison & Anderson),incorporated by reference.

By selecting for phenotypic characteristics among the cells obtainedfrom a solid tumor, solid tumor stem cells can be isolated from anyanimal solid tumor, particularly any mammalian solid tumor. It will beappreciated that, taking into consideration factors such as a bindingaffinities, that antibodies that recognize species-specific varieties ofmarkers are used to enrich for and select solid tumor stem cells.Antibodies that recognize the species-specific varieties of CD44, B38.1,CD24 and other markers will be used to enrich for or isolate solid tumorstem cells from that species (for example, antibody to a mouse CD44 formouse solid tumor stem cells, antibody to a monkey B38.1 for monkeysolid tumor stem cells, etc.).

An efficient xenograft model of human breast cancer. The inventionprovides a xenograft model in which to establish tumors by the injectionof solid tumor cells into a host animal. The host animal can be a modelorganism such as nematode, fruit fly, zebrafish; preferably a laboratorymammal such as a mouse (nude mouse, SCID mouse, NOD/SCID mouse,Beige/SCID Mouse), rat, rabbit, or primate. The severely immunodeficientNOD-SCID mice were chosen as recipients to maximize the participation ofinjected cells. Immunodeficient mice do not reject human tissues, andSCID and NOD-SCID mice have been used as hosts for in vivo studies ofhuman hematopoiesis and tissue engraftnent. McCune et al., Science 241:1632-9 (1988); Kamel-Reid & Dick, Science 242: 1706-9 (1988); Larochelleet al., Nat. Med. 2: 1329-37 (1996). In addition, Beige/SCID mice alsohave been used.

Xenograft tumors have been established from mastectomy specimens of allthe patients that have been tested to date (see, EXAMPLE 7). Tumors inmice have also been established from malignant pleural effusions. Inaddition, tumors have been established by the subcutaneous injection ofcells that have been obtained from two sarcomas. Furthermore, for allthe tumors that we have attempted, we have been able to make single-cellsuspensions (or suspensions with a few aggregates of cell, such as lessthan 100; preferably less than 10) and then transfer the tumors. Thisxenograft assay is useful for biological and molecular assays tocharacterize the tumorigenic, clonigenic solid tumor stem cells.

The NOD/SCID or Beige/SCID mice can be further immunosuppressed, usingVP-16 (see, EXAMPLES 1 and 3), radiation therapy, chemotherapy, or otherimmunosuppressive biological agents.

This in vivo assay is particularly advantageous for the betterunderstanding of breast cancer and development of new treatments forthis disease. Until now, it has been impossible to do biological andmolecular studies involving primary breast cancer. Such studies havebeen limited to cell lines. Unfortunately, it is well known that themany of the fundamental properties of breast cancer cells change intissue culture. Fenhall et al., British J. Cancer 81: 1142-1149 (1999).This latter problem only worsens with continued culturing of the cells.

By contrast, using the method of the invention, breast cancer cells(preferably enriched for breast cancer stem cells) are injected intoimmunocompromised mice, to grow the tumor. In one embodiment, the cellsare injected either into the mammary fat pads of mice or subcutaneouslyinto the mice. Furthermore, tumors can be established from single-cellsuspensions (or suspensions with a few aggregates of cell, such as lessthan 100; preferably less than 10) and then the tumors transferred toother mice.

The enrichment of solid tumor stem cells and the isolation of solidtumor stem cells distinguishes the present invention from the “primarybioassay of human tumor stem cells” referred to in U.S. Pat. No.4,411,990 (see also, Hamburger et al., Blood 47: 995 (1976); Salmon etal., AACR Abstracts 19: 231, Abstract No. 922 (1978)). In previoustissue culture assays, only a small proportion of the tumor cells wereable to form colonies in an in vitro clonogenic assay, and large numbersof cells (such as myeloma and hematopoietic cells) were typically neededto be transplanted to form tumors in vivo. Ogawa M et al., CancerResearch. 31(12): 2116-2119 (1971); Ogawa Metal., Cancer Research33(12): 3172-3175, 1973. Salmon S E & Hamburger A W, Science 197:461-463 (1977). Schlag P & Flentje D, Cancer Treatment Reviews 11 SupplA:131-7 (1984). This led to the hypothesis that only a small number oftumor cells are actually tumorigenic. However, because of technicallimitations, this tumorigenic fraction of cells could not be isolatedfrom non-tumorigenic cells and therefore it could not be proven thatthere were intrinsically different subsets of tumor cells, some withsubstantial proliferative potential and others with limited potential.That is, unless the tumorigenic cells can be purified and distinguishedfrom the non-tumorigenic cells it remains possible that all tumor cellshave a similar low probability of exhibiting clonogenic activity in anyassay. Moreover, without the ability to identify and isolate thetumorigenic fraction of cells (the tumor stem cells) U.S. Pat. No.4,411,990 lacks the utilities described in this invention. For example,without markers to isolate the tumorigenic cells it is not possible tostudy their gene expression patterns, or their expression of diagnosticmarkers, or their response to therapeutic agents. Several technicalproblems prevented prior inventions from isolating tumorigenic cells ortumor stem cells. First, in vitro assays resulted in some initial colonyformation, but usually the cells stopped proliferating and could not begrown continuously in culture. Salmon, S. E. & Hamburger A W, Science197:461463 (1977); Schlag P et al., Cancer Treatment Reviews. 11 SupplA:131-7, (1984); Salmon S E, Recent Results in Cancer Research 94: 8(1984). Also, cells from many tumors failed to form colonies in vitro atall. Carney D N et al, Stem Cells 1: 149-164 (1981). Similarly,dissociated cells isolated from most solid tumors rarely formed tumorsin immunodeficient mouse models. Sakakibara T et al., Cancer J, Si. Am2: 291-300 (1996); Mueller B & Reisfeld R A, Cancer Metastasis Rev. 10:193-200, (1991). The observation that only particular clones ofimmortalized tissue culture cancer cell lines were capable of formingtumors in the in vivo models further illustrates this problem (HamiltonT C et al., Cancer Research 44(11): 5286-90 (1984)). Thus, thelimitations in the assays made it impossible to determine whether thecolonies arose from stem cells that had lost their capacity toproliferate in vitro, from non-tumorigenic cells that had limitedproliferative potential, or whether the small number of cells able toform colonies in vitro was due to a “stem cell” population within thetumor or due to a rare cell that could proliferate in vitro.Furthermore, it was not possible to distinguish phenotypically differentpopulations of cells: prior to this invention, very limited use was madeof techniques like flow-cytometry to separate and analyze phenotypicallydistinct populations of solid tumor cells by flow-cytometry. Indeed, theclonogenic assays used in the prior art did not predict the behavior ofan individual patient's tumor and fell out of favor. Von Hoff D D etal., Cancer. 67(1): 20-7 (1991); Federico M et al., GynecologicOncology. 55(3 Pt 2): S156-63 (1994). Thus, the limitations in the cellseparation techniques, and the assays used in the prior art made itimpossible for them to purify tumorigenic cells. Therefore, it wasimpossible to prove the existence of hypothetical tumor stem cells.

Role of Notch in breast cancer. The Notch family of receptors has beenimplicated in stem cell development and differentiation (see, Morrisonet al., Cell 101(5): 499-510 (2000); Artavanis-Tsakonas et al., Science284: 770 (1999); and Artavanis-Tsakonas et al., Science 268: 225-232(1995); U.S. Pat. No. 6,090,922, incorporated by reference). Notch wasoriginally identified in Drosophila through loss-of-function mutationsthat produced too many neurons at the expense of other cell types.Poulson, Proc. Natl. Acad. Sci. USA 23: 133 (1937). In all animal modelstested, mutations in the Notch receptor result in developmentalabnormalities. In C. elegans, Notch is required for germ line stem cellself-renewal. Berry et al., Development 124(4): 925-36 (1997). In rats,Notch regulates neural crest stem cell differentiation. Morrison et al.,Cell 101(5): 499-510 (2000). Transient Notch activation initiates anirreversible switch from neurogenesis to gliogenesis by neural creststem cells.

Because neighboring cells can express Notch receptors and ligands, onecell can affect the fate of a neighboring cell by activating Notchsignaling in the neighboring cell.

Proteins with knife-edge names such as Jagged (Shimizu et al., Journalof Biological Chemistry 274(46) 32961-9 (1999); Jarriault et al.,Molecular and Cellular Biology 18: 7423-7431 (1998)), Serrate, and Delta(and variants of each, such as Delta1, Delta2, Delta3, Delta4, andJagged2, LAG-2 and APX-1 in C. elegans), bind to the Notch receptor andactivate a downstream signaling pathway that prevents neighboring cellsfrom becoming neural progenitors. A recently identified ligand is D114,a Notch ligand of the Delta family expressed in arterial endothelium.Shutter et al., Genes Dev 14(11): 1313-8 (2000)).

Notch ligands may bind and activate Notch family receptorspromiscuously. The expression of other genes, like Fringe family members(Panin et al, Nature 387(6636): 908-912 (1997)), may modify theinteractions of Notch receptors with Notch ligands. Numb family membersmay also modify Notch signaling intracellularly.

Ligand binding to Notch results in activation of apresenilin-1-dependent gamma-secretase-like protein that cleaves Notch.De Strooper et al., Nature 398: 518-522 (1999), Mumm et al., MolecularCell. 5: 197-206 (2000). Cleavage in the extracellular region mayinvolve a furin-like convertase. Logeat et al., Proceedings of theNational Academy of Sciences of the USA 95: 8108-8112 (1998). Theintracellular domain is released and transactivates genes by associatingwith the DNA binding protein RBP-J. Kato et al., Development 124:4133-4141 (1997)). Notch 1, Notch 2 and Notch 4 are thought totransactivate genes such as members of the Enhancer of Split (HES)family, while Notch 3 signaling may be inhibitory. Beatus et al.,Development 126: 3925-3935 (1999). Finally, secreted proteins in theFringe family bind to the Notch receptors and modify their function.Zhang & Gridley, Nature 394 (1998).

In mammals, there are four known Notch family members. Notch 4 is thehuman ortholog of the mouse int-3 oncogene that plays a role in breastcancer in mice. Gallahan et al., Cancer Res. 56(8): 1775-85 (1996);Uyttendaele et al., Development 2122: 251 (1996); Imatani & Callahan,Oncogene 19(2): 223-31 (2000)).

The invention is based upon the discovery that Notch 4 plays a role bothin normal human breast development and in tumorigenesis. Within anindividual tumor, only a small subpopulation of tumorigenic cellsexpresses high levels of Notch 4. An antibody that recognizes Notch 4blocks the growth of breast cancer tumor cells in vitro and in vivo(see, EXAMPLES 2, 5, 12 and 15). In one embodiment, the antibody bindsto the extracellular domain of Notch 4. In a particular embodiment, theantibody binds to the polypeptide region LLCVSVVRPRGLLCGSFPE(LeuLeuCysvalSerValValArgProArgGlyLeuLeuCysGly SerPheProGlu) (SEQ IDNO:19). However, any anti-Notch 4 antibody that inhibits Notchactivation can be used to impair tumor survival.

Inhibitors of Notch signaling (such as Numb and Numb-like; or antibodiesor small molecules that block Notch activation) can be used in themethods of the invention to inhibit solid tumor stem cells. In thismanner, the Notch pathway is modified to kill or inhibit theproliferation of solid tumor stem cells.

By contrast, it had previously been found that stimulation of Notchusing soluble Delta (Han et al., Blood 95(5): 161625 (2000)), a Notchligand, promoted growth and survival of tumor cells in vitro. Thus, ithad previously been found that stimulation of the Notch pathway promotesgrowth and survival of the cancer cells.

The invention differs from the manipulation of non-terminallydifferentiated cells using the Notch pathway provided in U.S. Pat. No.5,780,300. U.S. Pat. No. 5,780,300 addresses the modification of normalcells, not cancer cells. That patent is directed to methods for theexpansion of non-terminally differentiated cells (normal precursorcells) using agonists of Notch function, by inhibiting thedifferentiation of the cells without inhibiting proliferation (mitoticactivity) such that an expanded population of non-terminallydifferentiated cells is obtained. These expanded cells can be used incell replacement therapy, a use that is incompatible with the goal ofkilling or inhibiting the proliferation of solid tumor stem cells bymodifying Notch signaling in this invention.

Therapeutic aspects of the invention. A corollary to the solid tumorstem cell model of the invention is that, to effectively treat cancerand achieve higher cure rates, anti-cancer therapies must be directedagainst solid tumor stem cells. Since current therapies are directedagainst the bulk population, they may be ineffective at eradicatingsolid tumor stem cells. The limitations of current cancer therapiesderive from their inability to effectively kill solid tumor stem cells.The identification of solid tumor stem cells permits the specifictargeting of therapeutic agents to this cell population, resulting inmore effective cancer treatments. This concept would fundamentallychange our approach to cancer treatment.

Advances in modern biotechnology have facilitated the identification ofnew therapeutic targets for cancer treatment. Advances in genomics havemade it possible to sequence and identify the 10,000 to 30,000 genesthat are expressed in individual cell types. The human genome has beensequenced. This has resulted in the identification of new proteinsinvolved in a myriad of biological processes such as proliferation, celldeath and immortalization, providing targets for drug intervention.Although genomics provides a powerful means for identifying drug targetsin cancer cells, these targets are only valid if the targets are presentwithin the tumorigenic cell population. To be effective, genomics mustbe focused on individual populations within the heterogeneous cells thatcompose a tumor that are responsible for tumorigenic growth. In solidtumors, these are the solid tumor stem cells. Additionally, genomics hasnot yet been used to identify genes expressed in purified cellpopulations derived from cancerous tissues.

One of the major problems in identifying new cancer therapeutic agentsis determining which of the myriad of genes identified in large scalesequencing projects are the most clinically important drug targets. Thisis made especially difficult since solid tumors consist of a mixture ofa many types of normal cells and a heterogeneous population of tumorcells. One way to reduce the complexity is to make cDNA aftermicrodissection of solid tumors to enrich for tumor cells (see, below).This technique is based on the assumption that the pathologistdissecting out the tumor cells can predict which cells are tumorigenicbased upon appearance. However, cells can be morphologically similar andyet remain functionally heterogeneous. Moreover, cells obtained bymicrodissection are not viable and therefore the functional propertiesof such cells cannot be tested or verified.

Instead, by the methods of the invention, one can use flow cytometry(such as FACS) and the xenograft animal model of the invention to enrichfor specific cell populations. This technique has the advantage of beingable to simultaneously isolate phenotypically pure populations of viablenormal and tumor cells for molecular analysis. Thus, flow cytometryallows us to test the functions of the cell populations and use them inbiological assays in addition to studying their gene expressionprofiles. Furthermore, such cells can also be characterized inbiological assays. For example, mesenchymal (stromal) cells can beanalyzed for production of growth factors, matrix proteins andproteases, endothelial cells can be analyzed for production of specificfactors involved in solid tumor growth support (such asneo-vascularization), and different subsets of tumor cells from a solidtumor can be isolated and analyzed for tumorigenicity, drug resistanceand metastatic potential.

“Enriched”, as in an enriched population of cells, can be defined basedupon the increased number of cells having a particular marker in afractionated set of cells as compared with the number of cells havingthe marker in the unfractionated set of cells. However, the term“enriched can be preferably defined by tumorigenic function as theminimum number of cells that form tumors at limit dilution frequency intest mice. Thus, if 500 tumor stem cells form tumors in 63% of testanimals, but 5000 unfractionated tumor cells are required to form tumorsin 63% of test animals, then the solid tumor stem cell population is10-fold enriched for tumorigenic activity (see, EXAMPLES). The solidtumor stem cell model (FIG. 1A) provides the linkage between these twodefinitions of (phenotypic and functional) enrichment.

FACS methods using CD44 alone can enrich solid tumor stem cells at least2-fold (see, EXAMPLE 1 and 3). FACS methods using B38.1 and CD24 canenrich for solid tumor stem cells 5-6 fold (see, EXAMPLE 3). Enrichmentusing additional markers can enrich 10-fold or more and can be used toisolate solid tumor stem cells.

“Isolated” refers to a cell that is removed from its natural environment(such as in a solid tumor) and that is isolated or separated, and is atleast about 75% free, and most preferably about 90% free, from othercells with which it is naturally present, but which lack the markerbased on which the cells were isolated.

Purification (enrichment or isolation) of subsets of cancer cells from asolid tumor allows one of skill in the art of oncology to distinguishbetween classic models of cancers and the solid tumor stem cell model(FIG. 1). If indeed a minority of solid tumor cells has stem cellproperties, then to efficiently identify the genes necessary for tumorproliferation and drug resistance, the genomics must be focused on thestem cell population. If however, the genomics is targeted to the bulkpopulation rather than the solid tumor stem cells, then the mostpromising drug targets are obscured or lost in a sea of other genesexpressed by the other cells within a tumor that do not have thecapacity for extensive proliferation.

In some of the EXAMPLES, we focused on the tumorigenic cells from breastcancer. Focusing on the individual populations of cells within a solidtumor provides a clearer understanding of how to focus new cancertreatments and identify novel targets for drug discovery. In addition,purifying solid tumor stem (such as breast cancer tumorigenic) cellsprovides a material for screening for drug sensitivity and identifyingmarkers that predict tumorigenicity or metastatic potential.

In vivo proliferation of solid tumor stem cells. The in vivoproliferation of solid tumor stem cells can be accomplished by injectionof solid tumor stem cells into animals, preferably mammals, morepreferably in rodents such as mice (due to the predictable methods thathave been developed in the art for injection into laboratory rodents),and most preferably into immunocompromised mice, such as SCID mice,Beige/SCID mice or NOD/SCID mice (see, EXAMPLES). NOD/SCID mice areinjected with the varying number of cells and observed for tumorformation. The injection can be by any method known in the art,following the enrichment of the injected population of cells for solidtumor stem cells.

In one particular embodiment, to establish human breast cancer tumors inthe NOD/SCID mouse model, eight week old female NOD-SCID mice wereanesthetized by an intraperitoneal injection of 0.2 ml Ketamine/Xylazine(300 mg Ketamine combined with 20 mg Xylazine in a 4 ml volume. Then,0.02 ml of the solution was diluted in HBSS is used per 20 g mouse. Micewere then treated with VP-16 (etoposide) via an intraperitonealinjection (30 mg etoposide per 1 kg, diluted in serum-free HBSS for afinal injection volume of 0.2 ml). At the same time, estrogen pelletswere placed subcutanously on the back of the necks of the mice using atrocar. The mice were then warmed and placed back in to the cages afterthey awoke. All tumor injections/implantations were done 3-5 days afterthis procedure.

For the implantation of fresh specimens, samples of human breast tumorswere received within an hour after the surgeries. These tumors were cutup with scissors into small pieces, and the pieces were then minced witha blade to yield 2×2 mm-size pieces. Mincing was done in sterile RPMI1640 medium supplemented with 20% Fetal Bovine Serum under sterileconditions on ice. The tumor pieces were then washed with serum freeHBSS right before implantation. A 2-mm incision was then made in the midabdomen area, and using a trocar, one to two small tumor pieces wereimplanted onto the upper right and upper left mammary fat pats (rightbelow the second nipple on both sides). A 6-0 suture was wrapped twicearound the MFP-Nipple allowing it to hold the implanted pieces in place.Sutures were removed 5 days later. Nexaban was used to seal the incisionand mice were weekly monitored from tumor growth.

For the injection of the pleural effusions or dissociated solid tumorcells, cells were received shortly after surgery and washed with HBSSserum-free. Cells were then suspended in serum free-RPMI/Matrigelmixture (1:1 volume) and then injected into the upper right and leftmammary pads using an 18 G needle. To do this, the desired number ofcells were suspended in 0.2 ml and injected. The site of the needleinjection was sealed with Nexaban to prevent any cell leakage.

For the injection of digested tumor cells, tumors from a patient (solidtumors) or grown in mice (by the methods of the invention) were cut upinto small pieces and then minced completely using sterile blades. Theresulting pieces were then mixed with ultra-pure Collagenase III in HBSSsolution (200-250 U collagenase/ml) and allowed to incubate at 37 C for3-4 hr, pipetting with a 10 ml pipette is done every 15-20 minutes. Atthe end of the incubation, cells were filtered through a 45-micron nylonmesh and washed with RPMI-20% FBS, then washed with HBSS twice. Cells tobe injected were then suspended in HBSS/Matrigel mix (1:1 volume) andinjected into the mammalian fat pad or subcutaneously as describedabove. Nexaban can be used to seal the injection site.

For analysis of the xenotransplant tumor, a solid tumor is removed fromthe mice and made into a single cell suspension. Cells are stained andanalyzed by flow cytometry (FACS) using methods known to those skilledin the art (Morrison & Weissman, Immunity 1(8): 661-73 (1994)). Thephenotype of tumorigenic cells is CD44⁺CD24^(−/lo) in all tumors, andB38.1⁺CD44⁺CD24^(−/lo) in most tumors. We then do limiting dilutionanalysis of cells isolated by FACS based upon expression of thesemarkers. Next, we further purify the breast cancer stem cell. Cells arestained with 7AAD (which stains dead cells), anti H2K-PE (which stainsmouse cells), and combinations of antibodies against various markersthat have heterogeneous expression patterns by the cancer cellsincluding anti-B38.1, -annexin V, -Notch 4, -CD9, -CD24, -MUC1, -CD49F,-CD62P, -P-glycoprotein, -Notch 1, -520C9, -260F9 and -317G5. FACS isused to isolate viable human cells that either do or do not express oneof the differentially expressed antigens. A combination of markersallows the greatest enrichment of tumorigenic cells. For the limitingdilution assays, one hundred, one thousand, ten thousand and one hundredthousand cells of each population are analyzed in vivo.

SCID mice, NOD/SCID mice or Beige/SCID mice are injected with thevarying number of cells and observed for tumors. Any tumors that formare removed for pathologic examination and FACS analysis. The tests arerepeated (for example, about ten times) to confirm the results. Thephenotypes of the tumorigenic cells are thus determined.

Other general techniques for formulation and injection of cells may befound in Remington's Pharmaceutical Sciences, 20th ed. (Mack PublishingCo., Easton, Pa.). Suitable routes may include parenteral delivery,including intramuscular, subcutaneous (see, above), intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections,just to name a few. For injection, the agents of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. For such transmucosal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

By the use of populations of cells enriched for solid tumor stem cells,the invention is an improvement over the methods of Mueller & Reisfeld,Cancer Metastasis Rev. 10: 193-200 (1991) (who used the SCID mouse,which allows disseminated growths for a number of human tumors,particularly hematologic disorders and malignant melanoma) andSakakibara et al., Cancer J. Sci. Am. 2: 291-300 (1996) (who studied thegrowth and metastatic potential of surgical specimens of breastcarcinomas engrafted into the large abdominal (gonadal) fat pad ofsevere combined immunodeficient (SCID) mice). Sakakibara et al. observedthat placement of human breast tumors within the gonadal fat pad couldresult in tumors that grew either rapidly, slowly, or not at all. Of 48tumors studied, 12 (25%), including one of the three lymph node-derivedtumors, grew rapidly enough within some or all of the implanted mice(i.e., the tumors reached a diameter of 2-3 cm within 2-6 months) toallow repeated passage.

By contrast, the injection of solid tumor stem cells can consistentlyresult in the successful establishment of tumors, more than 75% of thetime, preferably more than 80% of the time, more preferably more than85%, more than 90%, or more than 95% of the time. We have achieved 100%successful establishment of tumors from the five tumors tested, as wellas from three pleural efflusions (see, EXAMPLES). Moreover, theinvention provides for the advantageous establishment of solid tumors(particularly tumors from breast tumor stem cells) in mammary fat pads,an area not accessable for establishment using the methods of Sakakibaraet al., Cancer J. 2: 291-300 (1996).

In vitro proliferation of solid tumor stem cells. Cells can be obtainedfrom solid tumor tissue by dissociation of individual cells. Tissue froma particular tumor is removed using a sterile procedure, and the cellsare dissociated using any method known in the art (see, Sambrook et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1989); Current Protocols in Molecular Biology,Ausubel et al., eds., (Wiley Interscience, New York, 1993), andMolecular Biology LabFax, Brown, ed. (Academic Press, 1991)), includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrument.Methods of dissociation are optimized by testing differentconcentrations of enzymes and for different periods of time, to maximizecell viability, retention of cell surface markers, and the ability tosurvive in culture (Worthington Enzyme Manual, Von Worthington, ed.(Worthington Biochemical Corporation, 2000). Dissociated cells arecentrifuged at low speed, between 200 and 2000 rpm, usually about 1000rpm (210 g), and then resuspended in culture medium. For guidance tomethods for cell culture, see Spector et al., Cells: A Laboratory Manual(Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1998).

The dissociated tumor cells can be placed into any known culture mediumcapable of supporting cell growth, including EM, DMEM, RPMI, F-12, andthe like, containing supplements which are required for cellularmetabolism such as glutamine and other amino acids, vitamins, mineralsand useful proteins such as transferrin and the like. Medium may alsocontain antibiotics to prevent contamination with yeast, bacteria andfungi such as penicillin, streptomycin, gentamicin and the like. In somecases, the medium may contain serum derived from bovine, equine, chickenand the like. However, a preferred embodiment for proliferation of solidtumor stem cells is to use a defined, low-serum culture medium. Apreferred culture medium for solid tumor stem cells is a defined culturemedium comprising a mixture of Ham's F12, 2% fetal calf serum, and adefined hormone and salt mixture, either insulin, transferrin, andselenium or B27 supplement. Brewer et al, J. Neuroscience Res. 35: 567(1993).

The culture medium can be a chemically defined medium that issupplemented with fetal bovine serum or chick embryo extract (CEE) as asource of mitogens and survival factors to allow the growth of tumorstem cells in culture. Other serum-free culture medium containing one ormore predetermined growth factors effective for inducing stem cellproliferation, such as N2 supplement or B27 supplement, known to thoseof skill in the art can be used to isolate and propagate solid tumorstem cells from other bird and mammalian species, such as human. See,U.S. Pat. Nos. 5,750,376, 5,851,832, and 5,753,506; Atlas et al.,Handbook of Microbiological Media (CRC Press, Boca, Raton, La., 1993);Freshney, Cutler on Animal Cells, A Manual of Basic Technique, 3dEdition (Wiley-Liss, New York, 1994), all incorporated herein byreference.

The culture medium for the proliferation of solid tumor stem cells thussupports the growth of solid tumor stem cells and the proliferatedprogeny. The “proliferated progeny” are undifferentiated tumor cells,including solid tumor stem cells, since solid tumor stem cells have acapability for extensive proliferation in culture.

Conditions for culturing should be close to physiological conditions.The pH of the culture medium should be close to physiological pH,preferably between pH 6-8, more preferably between about pH 7 to 7.8,with pH 7.4 being most preferred. Physiological temperatures rangebetween about 30° C. to 40° C. Cells are preferably cultured attemperatures between about 32° C. to about 38° C., and more preferablybetween about 35° C. to about 37° C. Similarly, cells may be cultured inlevels of O₂ that are comparatively reduced relative to O.sub.2concentrations in air, such that the O₂ concentration is comparable tophysiological levels (1-6%), rather than 20% O₂ in air.

A particular patient's solid tumor stem cells; once they have beenproliferated in vitro, can be analyzed and screened. Solid tumor stemcell proliferated in vitro can also be genetically modified usingtechniques known in the art (see, below; see also, Sambrook et al.,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1989); Current Protocols in Molecular Biology,Ausubel et al., eds., (Wiley Interscience, New York, 1993)). The invitro genetic modification may be more desirable in certaincircumstances than in vivo genetic modification techniques when morecontrol over the infection with the genetic material is required.

Solid tumor stem cells and stem cell progeny can be cryopreserved untilthey are needed by any method known in the art. The cells can besuspended in an isotonic solution, preferably a cell culture medium,containing a particular cryopreservant. Such cryopreservants includedimethyl sulfoxide (DMSO), glycerol and the like. These cryopreservantsare used at a concentration of 5-15%, preferably 8-10%. Cells are frozengradually to a temperature of −10° C. to −150° C., preferably −20° C. to−100° C., and more preferably −150° C.

Additional guidance for the in vitro culture of solid tumor stem cellsis provided in EXAMPLE 9 and FIG. 9.

Genetic modification of solid tumor stem cells and solid tumor stem cellprogeny. In the undifferentiated state, the solid tumor stem cellsrapidly divide and are therefore excellent targets for geneticmodification. The term “genetic modification” as used herein refers tothe stable or transient alteration of the genotype of a precursor cellby intentional introduction of exogenous DNA. DNA may be synthetic, ornaturally derived, and may contain genes, portions of genes, or otheruseful DNA sequences. The term “genetic modification” as used herein isnot meant to include naturally occurring alterations such as that whichoccurs through natural viral activity, natural genetic recombination, orthe like. General methods for the genetic modification of eukaryoticcells are known in the art. See, Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,1989); Current Protocols in Molecular Biology, Ausubel et al., eds.,(Wiley Interscience, New York, 1993)).

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use with solid tumor stem cells in vivo, invitro, and ex vivo. Vectors may be introduced into hematopoietic stemcells taken from the patient and clonally propagated. By the method ofthe invention, such methods are extended to solid tumor stem cells.

“Transformation,” or “genetically modified” as defined herein, describesa process by which exogenous DNA enters and changes a recipient cell.Transformation may occur under natural or artificial conditionsaccording to various methods well known in the art, and may rely on anyknown method for the insertion of foreign nucleic acid sequences into aprokaryotic or eukaryotic host cell. The method for transformation isselected based on the type of host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation, heatshock, lipofection, and particle bombardment. The term “transformed”cells includes stably transformed cells in which the inserted DNA iscapable of replication either as an autonomously replicating plasmid oras part of the host chromosome, as well as transiently transformed cellswhich express the inserted DNA or RNA for limited periods of time.

Genetic manipulation of primary tumor cells has been describedpreviously by Patel et al., Human Gene Therapy 5: 577-584 (1994).Genetic modification of a cell may be accomplished using one or moretechniques well known in the gene therapy field. Mulligan R C, HumanGene Therapy 5: 543-563 (1993). Viral transduction methods may comprisethe use of a recombinant DNA or an RNA virus comprising a nucleic acidsequence that drives or inhibits expression of a protein to infect atarget cell. A suitable DNA virus for use in the present inventionincludes but is not limited to an adenovirus (Ad), adeno-associatedvirus (AAV), herpes virus, vaccinia virus or a polio virus. A suitableRNA virus for use in the present invention includes but is not limitedto a retrovirus or Sindbis virus. Several such DNA and RNA viruses existthat may be suitable for use in the present invention.

Adenoviral vectors have proven especially useful for gene transfer intoeukaryotic cells for vaccine development (Graham F L & Prevec L, InVaccines: New Approaches to Immunological Problems, Ellis R V ed.,363-390 (ButterWorth-Heinemann, Boston, 1992).

Specific guidance for the genetic modification of solid tumor stem cellsis provides in EXAMPLE 13 and in FIGS. 15-18.

“Non-viral” delivery techniques that have been used or proposed for genetherapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes,direct injection of DNA, CaPO₄ precipitation, gene gun techniques,electroporation, and lipofection. Mulligan R C, Science 260: 926-932(1993). Any of these methods are widely available to one skilled in theart and would be suitable for use in the present invention. Othersuitable methods are available to one skilled in the art, and it is tobe understood that the present invention may be accomplished using anyof the available methods of transfection. Lipofection may beaccomplished by encapsulating an isolated DNA molecule within aliposomal particle and contacting the liposomal particle with the cellmembrane of the target cell. Liposomes are self-assembling, colloidalparticles in which a lipid bilayer, composed of amphiphilic moleculessuch as phosphatidyl serine or phosphatidyl choline, encapsulates aportion of the surrounding media such that the lipid bilayer surrounds ahydrophilic interior. Unilammellar or multilammellar liposomes can beconstructed such that the interior contains a desired chemical, drug,or, as in the instant invention, an isolated DNA molecule. Delivery bytransfection, by liposome injections, or by polycationic amino polymersmay be achieved using methods which are well known in the art (see,e.g., Goldman, C. K. et al. Nature Biotechnology 15:462-466 (1997)).

Two types of modified solid tumor stem cells of particular interest aredeletion mutants and over-expression mutants. Deletion mutants arewild-type cells that have been modified genetically so that a singlegene, usually a protein-coding gene, is substantially deleted. Deletionmutants also include mutants in which a gene has been disrupted so thatusually no detectable mRNA or bioactive protein is expressed from thegene, even though some portion of the genetic material may be present.In addition, in some embodiments, mutants with a deletion or mutationthat removes or inactivates one activity of a protein (oftencorresponding to a protein domain) that has two or more activities, areused and are encompassed in the term “deletion mutants.” Over-expressionmutants are wild-type cells that are modified genetically so that atleast one gene, most often only one, in the modified solid tumor stemcell is expressed at a higher level as compared to a cell in which thegene is not modified.

Genetically modified solid tumor stem cells can be subjected to tissueculture protocols known in the art (see, U.S. Pat. Nos. 5,750,376 and5,851,832, Spector et al., Cells: A Laboratory Manual (Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1998)). Tumor stem cells can begenetically modified in culture to promote differentiation, cell death,or immunogenicity. For example, tumor stem cells can be modified toenhance expression of products that direct an immune response againstthe patient's solid tumor. Alternatively, the solid tumor stem cells canbe subjected to various proliferation protocols in vitro prior togenetic modification. The protocol used depends upon the type ofgenetically modified solid tumor stem cell or solid tumor stem cellprogeny desired. Once the cells have been subjected to thedifferentiation protocol, they are again assayed for expression of thedesired protein. Cells having the desired phenotype can be isolated andimplanted into recipients in need of the protein or biologically activemolecule that is expressed by the genetically modified cell. Suchmolecules can enhance tumor regression or inhibit tumor spread.

In vitro models of solid tumor development, in vivo models, and methodsfor screening effects of drugs on solid tumor stem cells. Solid tumorstem cells and solid tumor stem cell progeny cultured in vitro (see,EXAMPLE 9) or in vivo (in the xenograft model of the invention) can beused for the screening of potential therapeutic compositions. Thesecompositions for the treatment of solid tumors can be applied to thesecells in culture at varying dosages, and the response of these cellsmonitored for various time periods. Physical characteristics of thesecells can be analyzed by observing cells by microscopy. The induction ofexpression of new or increased levels of proteins such as enzymes,receptors and other cell surface molecules can be analyzed with anytechnique known in the art see, Clarke et al., Proc. Natl. Acad. Sci.USA 92: 11024-11028 (1995)) which can identify the alteration of thelevel of such molecules. These techniques include immunohistochemistry,using antibodies (see, EXAMPLES) against such molecules, or biochemicalanalysis. Such biochemical analysis includes protein assays, enzymaticassays, receptor binding assays, enzyme-linked immunosorbant assays(ELISA), electrophoretic analysis, analysis with high performance liquidchromatography (HPLC), Western blots, and radioimmune assays (RIA).Nucleic acid analysis such as Northern blots can be used to examine thelevels of mRNA coding for these molecules or PCR (see, EXAMPLE 14).

Alternatively, such cells treated with these pharmaceutical compositionscan be transplanted into an animal (such as in the xenograft model ofthe invention), and their survival, ability to form tumors, andbiochemical and immunological characteristics examined.

The solid stem cells and solid stem cell progeny of the invention can beused in methods of determining the effect of a biological agents onsolid tumor cells. The term “biological agent” or “test compound” refersto any agent (including a virus, protein, peptide, amino acid, lipid,carbohydrate, nucleic acid, nucleotide, drug, antibody, prodrug or othersubstance) that may have an effect on tumor cells whether such effect isharmful, beneficial, or otherwise.

To determine the effect of a potential test compound on solid tumor stemcells, a culture of precursor cells derived from tumor stem cells can beobtained from tissue of a subject, such as a patient with a tumor orother cancerous disease, such as an epithelial cancer or breast cancer.Once the solid tumor stem cells or other desired populations of cellsare obtained from the subject, the cells are cultured in vitro. Thechoice of culture depends upon the particular test compound being testedand the diagnostic effects that the laboratory personnel want toachieve.

The ability of various biological agents to increase, decrease, ormodify in some other way the number and nature of the solid tumor stemcells and solid tumor stem cell progeny can be screened. For example, itis possible to screen for test compounds that decrease the proliferativeability of the solid tumor stem cells, which would be useful foridentifying anti-cancer therapeutic agents. In these assays, therelevant cells are cultured in the presence of the test compounds ofinterest and assayed for the degree of proliferation or cell death thatoccurs.

The effects of a test compound or combination of test compounds on theextensive proliferation of solid tumor stem cells and their progeny canbe determined. It is possible to screen non-tumorigenic solid tumorcells that have already been induced to lose the ability to extensivelyproliferate before the screening. It is also possible to determine theeffects of the test compounds on the proliferation process by applyingthem to solid tumor stem cells. Generally, the test compound issolubilized and added to the culture medium or to the mouse in the invivo assay at varying concentrations to determine the effect of the testcompounds or agent at each dose. The culture medium may be replenishedwith the test compound or biological agent every couple of days inamounts so as to keep the concentration of the agent somewhat constant.Similarly, the test compound can be re-administered to the mouse atdifferent intervals to assess the effect of the compound over time.

Changes in proliferation are observed by an increase or decrease in thenumber of cells that form or an increase or decrease in the size of thefoci in vitro, or tumor size in vivo (which is a reflection of the rateof proliferation and the rate of cell death—determined by the numbers ofcells per foci or tumor size in the mouse). The effect of the testcompound on tumor stem cells are measured by determining the number oftumor stem cells that persist in culture or in the tumors in vivo aftertreatment with the test compound. In addition to determining the numberof tumor stem cells, the effects of the test compound on tumor stem cellcell-cycle status, and marker expression are also determined byflow-cytometry.

The test compounds or biological agents added to the culture medium orinjected into the mouse can be at a final concentration in the range ofabout 10 pg/ml to 1 μg/ml, preferably about 1 ng/ml (or 1 ng/cc ofblood) to 100 ng/ml (or 100 ng/cc of blood).

The effects of the test compounds or biological agents are identified onthe basis of significant difference relative to control cultures withrespect to criteria such as the ratios of expressed phenotypes, cellviability, proliferation rate, number of tumor stem cells, tumor stemcell activity upon transplantation in vivo, tumor stem cell activityupon transplantation in culture, cell cycle distribution of tumor cells,and alterations in gene expression.

Therapeutic compositions and methods. A pharmaceutical compositioncontaining a Notch ligand, an anti-Notch antibody, or other therapeuticagent that acts as an agonist or antagonist of proteins in the Notchsignal transduction/response pathway can be administered by anyeffective method. For example, a physiologically appropriate solutioncontaining an effective concentration of anti-Notch therapeutic agentcan be administered topically, intraocularly, parenterally, orally,intranasally, intravenously, intramuscularly, subcutaneously or by anyother effective means. In particular, the anti-Notch therapeutic agentmay be directly injected into a target cancer or tumor tissue by aneedle in amounts effective to treat the tumor cells of the targettissue. Alternatively, a cancer or tumor present in a body cavity suchas in the eye, gastrointestinal tract, genitourinary tract (e.g., theurinary bladder), pulmonary and bronchial system and the like canreceive a physiologically appropriate composition (e.g., a solution suchas a saline or phosphate buffer, a suspension, or an emulsion, which issterile) containing an effective concentration of anti-Notch therapeuticagent via direct injection with a needle or via a catheter or otherdelivery tube placed into the cancer or tumor afflicted hollow organ.Any effective imaging device such as X-ray, sonogram, or fiber-opticvisualization system may be used to locate the target tissue and guidethe needle or catheter tube. In another alternative, a physiologicallyappropriate solution containing an effective concentration of anti-Notchtherapeutic agent can be administered systemically into the bloodcirculation to treat a cancer or tumor that cannot be directly reachedor anatomically isolated.

All such manipulations have in common the goal of placing the anti-Notchtherapeutic agent in sufficient contact with the target tumor to permitthe anti-Notch therapeutic agent to contact, transduce or transfect thetumor cells (depending on the nature of the agent). In one embodiment,solid tumors present in the epithelial linings of hollow organs may betreated by infusing the vector suspension into a hollow fluid filledorgan, or by spraying or misting into a hollow air filled organ. Thus,the tumor cells (such as a solid tumor stem cells) may be present in oramong the epithelial tissue in the lining of pulmonary bronchial tree,the lining of the gastrointestinal tract, the lining of the femalereproductive tract, genitourinary tract, bladder, the gall bladder andany other organ tissue accessible to contact with the anti-Notchtherapeutic agent. In another embodiment, the solid tumor may be locatedin or on the lining of the central nervous system, such as, for example,the spinal cord, spinal roots or brain, so that anti-Notch therapeuticagent infused in the cerebrospinal fluid contacts and transduces thecells of the solid tumor in that space. (Accordingly, the anti-Notchtherapeutic agent can be modified to cross the blood brain barrier usingmethod known in the art). One skilled in the art of oncology canappreciate that the anti-Notch therapeutic agent can be administered tothe solid tumor by direct injection of the vector suspension into thetumor so that anti-Notch therapeutic agent contacts and affects thetumor cells inside the tumor.

One skilled in the art of oncology can understand that the vector isadministered in a composition comprising the vector together with acarrier or vehicle suitable for maintaining the transduction ortransfection efficiency of the chosen vector and promoting a safeinfusion. Such a carrier may be a pH balanced physiological buffer, suchas a phosphate, citrate or bicarbonate buffers a saline solution, a slowrelease composition and any other substance useful for safely andeffectively placing the anti-Notch therapeutic agent in contact withsolid tumor stem cells to be treated.

In treating a cancer patient who has a solid tumor, a therapeuticallyeffective amount of an anti-Notch therapeutic agent is administered. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms or a prolongation ofsurvival in a patient. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD5₅₀0/ED₅₀50. Compounds that exhibitlarge therapeutic indices are preferred. The data obtained from thesecell culture assays and animal studies can be used in formulating arange of dosage for use in humans. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography (HPLC).

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (see e.g.Fingl et al., In The Pharmacological Basis of Therapeutics, Ch. 1, pg. 1(1975)). The attending physician would know how to and when toterminate, interrupt, or adjust administration due to toxicity, or toorgan dysfunctions. Conversely, the attending physician would also knowto adjust treatment to higher levels if the clinical response were notadequate (precluding toxicity). The magnitude of an administrated dosein the management of the clinical disorder of interest can vary with theseverity of the condition to be treated and the route of administration.See, Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals,12 Edition (CRC Press 1996); Physicians' Desk Reference 55^(th) Edition(2000)). The severity of the condition may, for example, be evaluated,in part, by appropriate prognostic evaluation methods. Further, the doseand perhaps dose frequency, also vary according to the age, body weight,and response of the individual patient. A program comparable to thatdiscussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 20th ed. (Mack Publishing Co., Easton, Pa.).Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHank's solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, capsules, or solutions. The pharmaceutical compositions of thepresent invention may be manufactured in a manner that is itself known,e.g., by means of conventional mixing, dissolving, granulating,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Polynucleotides and polypeptides obtained from solid tumor stem cells.“Polynucleotide” refers to chain of nucleotides, which can be a nucleicacid, nucleic acid sequence, oligonucleotide, nucleotide, or anyfragment thereof. It may be DNA or RNA of genomic DNA, mRNA, cDNA, orsynthetic origin, double-stranded or single-stranded, and combined withcarbohydrate, lipids, protein or other materials to perform a particularactivity or form a useful composition. “Oligonucleotide” issubstantially equivalent to the terms amplimer, primer, oligomer,element, and probe. The term “probe” refers to a polynucleotide sequencecapable of hybridizing with a target sequence to form a polynucleotideprobe/target complex. A “target polynucleotide” refers to a chain ofnucleotides to which a polynucleotide probe can hybridize by basepairing. In some instances, the sequences will be complementary (nomismatches) when aligned. In other instances, there may be up to a 10%mismatch.

DNA or RNA can be isolated from the sample according to any of a numberof methods well known to those of skill in the art. For example, methodsof purification of nucleic acids are described in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology: Hybridization WithNucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation(Elsevier, New York N.Y., 1993). “Sample” is used in its broadest sense.A sample containing polynucleotides or polypeptides can be a bodilyfluid; an extract from a cell, chromosome, organelle, or membraneisolated from a cell; genomic DNA, RNA, or cDNA in solution or bound toa substrate; a cell; a tissue; a tissue print; and the like.

Total RNA can be isolated using the TRIZOL reagent (Life Technologies,Gaithersburg Md., USA), and mRNA is isolated using oligo d(T) columnchromatography or glass beads. Alternatively, when targetpolynucleotides are derived from an mRNA, the target polynucleotides canbe a cDNA reverse transcribed from an mRNA, an RNA transcribed from thatcDNA, a DNA amplified from that cDNA, an RNA transcribed from theamplified DNA, and the like. When the target polynucleotide is derivedfrom DNA, the target polynucleotide can be DNA amplified from DNA or RNAreverse transcribed from DNA.

Several technologies produce pools of restriction fragments of limitedcomplexity for electrophoretic analysis, such as methods combiningdouble restriction enzyme digestion with phasing primers (see, e.g.,European patent application EP 0 534 858 A1), or methods selectingrestriction fragments with sites closest to a defined mRNA end (see,e.g., Prashar et al., Proc. Natl. Acad. Sci. USA 93: 659-663 (1996)).Other methods statistically sample cDNA pools, such as by sequencingsufficient bases (in each of multiple cDNAs to identify each cDNA, or bysequencing short tags which are generated at known positions relative toa defined mRNA end (see, e.g., Velculescu, Science 270: 484-487 (1995)).

Methods of modifying RNA abundances and activities currently fall withinthree classes, ribozymes, antisense species (PCT patent application WO88/09810), and RNA aptamers (Good et al., Gene Therapy 4: 45-54 (1997)).Ribozymes are RNAs which are capable of catalyzing RNA cleavagereactions. (PCT patent application WO 90/11364). Ribozyme methodsinvolve exposing a cell to, inducing expression in a cell, etc. of suchsmall RNA ribozyme molecules. (Grassi & Marini, Annals of Medicine 28:499-510 (1996); Gibson, Cancer and Metastasis Reviews 15: 287-299(1996)).

The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to the “sense”strand of a specific nucleic acid sequence. Antisense molecules may beproduced by any method including synthesis or transcription. Onceintroduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes and to blockeither transcription or translation. The designation “negative” canrefer to the antisense strand, and the designation “positive” can referto the sense strand.

Oligonucleotides may be synthesized by standard methods known in theart, e.g. by use of an automated DNA synthesizer (such as arecommercially available from Biosearch, Applied Biosystems, etc.).

“Amplification,” as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. See, e.g., Dieffenbach C W & Dveksler G S, PCR Primer, a LaboratoryManual 1-5(Cold Spring Harbor Press, Plainview, N.Y., 1995).Amplification can be polymerase chain reaction (PCR), ligase chainreaction (LCR), nucleic acid sequence-based amplification (NASBA), or T7based RNA amplification.

“Polypeptide” refers to an amino acid, amino acid sequence,oligopeptide, peptide, or protein or portions thereof whether naturallyoccurring or synthetic.

Methods for direct measurement of protein activity are well known tothose of skill in the art. Such methods include, e.g., methods whichdepend on having an antibody ligand for the protein, such as Westernblotting, see, e.g., Burnette, A. Anal. Biochem. 112: 195-203 (1981).Such methods also include enzymatic activity assays, which are availablefor most well-studied protein drug targets. Detection of proteins can beaccomplished by antibodies (see, EXAMPLES).

The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

Proteins isolated from an enriched population of solid tumor stem cellsor isolated solid tumor cells can be separated by two-dimensional gelelectrophoresis systems. Two-dimensional gel electrophoresis iswell-known in the art and typically involves iso-electric focusing alonga first dimension followed by SDS-PAGE electrophoresis along a seconddimension. See, e.g., Hames et al, Gel Electrophoresis of Proteins: APractical Approach (IRL Press, New York 1990); Lander, Science274:536-539 (1996). The resulting electrophoretograms can be analyzed bynumerous techniques, including mass spectrometric techniques, westernblotting and immunoblot analysis using polyclonal and monoclonalantibodies, and internal and N-terminal micro-sequencing. Using thesetechniques, it is possible to identify a substantial fraction of all theproteins produced under given physiological conditions, including incells (e.g., in solid tumor stem cells) exposed to a drug, or in cellsmodified by, e.g., deletion or over-expression of a specific gene.

cDNA libraries. The purified solid tumor stem cells, solid tumor stemcell progeny, non-tumorigenic cells, and unfractionated tumor cells canbe used to make arrays or cDNA libraries using methods known in the art(see, Sambrook et al., Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Press, Cold Spring Harbor, N.Y., 1989); Current Protocolsin Molecular Biology, Ausubel et al., eds., (Wiley Interscience, NewYork, 1993)) to identify potential novel drug targets.

Molecular biology comprises a wide variety of techniques for theanalysis of nucleic acids and proteins, many of which form the basis ofclinical diagnostic assays. These techniques include nucleic acidhybridization analysis, restriction enzyme analysis, genetic sequenceanalysis, and separation and purification of nucleic acids and proteins(Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed.(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).Many molecular biology techniques involve carrying out numerousoperations on a large number of samples. For guidance to genomics andother molecular biological methods useful in the invention, see Birrenet al., Genome Analysis: A Laboratory Manual Series, Volume 1, AnalyzingDNA (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1997); Birrenet al., Genome Analysis: A Laboratory Manual Series, Volume 2, DetectingGenes (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1998); Birrenet al., Genome Analysis: A Laboratory Manual Series, Volume 4, MappingGenomes (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1999).

Nucleic acid hybridization analysis generally involves the detection ofa very small numbers of specific target nucleic acids (DNA or RNA) withprobes among a large amount of non-target nucleic acids. Multiple samplenucleic acid hybridization analysis has been conducted on a variety offilter and solid support formats. The “dot blot” hybridization, involvesthe non-covalent attachment of target DNAs to a filter, which aresubsequently hybridized with a labeled probes. The “dot blot”hybridization has been further developed for multiple analysis (EuropeanPatent application EP 0 228 075) and for the detection of overlappingclones and the construction of genomic maps (U.S. Pat. No. 5,219,726).Another format, the so-called “sandwich” hybridization, involvesattaching oligonucleotide probes covalently to a solid support and usingthem to capture and detect multiple nucleic acid targets (U.S. Pat. No.4,751,177; PCT International patent application WO 90/01564). Multiplexversions of these formats are called “reverse dot blots”.

Methods are known in the art for amplifying signal using sensitivereporter groups (enzyme, fluorophores, radioisotopes, etc.) andassociated detection systems (fluorometers, luminometers, photoncounters, scintillation counters, etc.). These methods can be combinedwith amplification methods, such as the polymerase chain reaction (PCR)for the amplification of target nucleic acid sequences. See, Innis etal., PCR Protocols: A Guide to Methods and Applications, (AcademicPress, 1990).

Microarrays. Mimicking the in situ hybridization in some aspects, newtechniques are being developed for carrying out multiple sample nucleicacid hybridization analysis on micro-formatted multiplex or matrixdevices (e.g., DNA chips) (see, Barinaga, Science 253: 1489 (1991);Bains, Bio/Technology 10: 757-758 (1992)). Guidance for the use ofmicroarrays is provided by Wang, E et al., Nature Biotechnology 18;457-459 (2000); Diehn M et al., Nature Genetics 25: 58-62 (2000).

Microarrays are known in the art and consist of a surface to whichprobes that correspond in sequence to gene products (e.g., cDNAs,oligonucleotides, mRNAs, cRNAs, polypeptides, and fragments thereof),can be specifically hybridized or bound at a known position. In oneembodiment, the microarray is an array (i.e., a matrix) in which eachposition represents a discrete binding site for a product encoded by agene (e.g., a protein or RNA), and in which binding sites are presentfor products of most or almost all of the genes in the organism'sgenome.

The polynucleotides, polypeptides, or analogues are attached to a solidsupport or substrate, which may be made from glass, plastic (e.g.,polypropylene, nylon), polyacrylamide, nitrocellulose, or othermaterials. “Substrate” refers to any suitable rigid or semi-rigidsupport to which polynucleotides or polypeptides are bound and includesmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, capillaries or other tubing, plates, polymers,and microparticles with a variety of surface forms including wells,trenches, pins, channels and pores. The polynucleotides can beimmobilized on a substrate by any method known in the art. Preferably,the substrates are optically transparent.

A variety of methods are currently available for making arrays ofbiological macromolecules, such as arrays of nucleic acid molecules orproteins. One method for making ordered arrays of DNA on a porousmembrane is a “dot blot” or “slot-blot” method. A more efficienttechnique employed for making ordered arrays of fragments uses an arrayof pins dipped into the wells, e.g., the 96 wells of a microtiter plate,for transferring an array of samples to a substrate, such as a porousmembrane. An alternate method of creating ordered arrays of nucleic acidsequences is described by U.S. Pat. No. 5,143,854 (to Pirrung) and alsoby Fodor et al., Science 251:767-773 (1991). The method involvessynthesizing different nucleic acid sequences at different discreteregions of a support. Khrapko et al., DNA Sequence 1:375-388 (1991)describes a method of making an oligonucleotide matrix by spotting DNAonto a thin layer of polyacrylamide, manually with a micropipette. U.S.Pat. No. 5,807,522 (to Brown et al., incorporated by reference)describes methods for fabricating microarrays of biological samples bydispensing a known volume of a reagent at each selected array position,by tapping a capillary dispenser on the support under conditionseffective to draw a defined volume of liquid onto the support.

Spotters can use pin, ink-jet, and other technologies to deposit samplesonto the support material. Several of the more common methods utilizemetal pins, which can be either solid or split. When the pins are dippedinto wells that contain the compounds of interest, each picks up a smallamount of the material. The pin is then brought into contact with thesolid support and a nanoliter volume is dispensed at the desiredlocation. In split pins (otherwise known as quills) a slot cut into thehead of the pin functions as a reservoir for the compound being spotted.Quill pins are most often used with glass slides, while solid pins aretypically used for spotting membranes. Amersham Pharmacia Biotech,GeneMachines, and other companies offer spotting robots.

Ink-jet technology provides another method of spotting microarrays.Adapted from the printer industry and redesigned for use inbiotechnological applications, this uses piezoelectric crystaloscillators and an electrode guidance system to deposit the compound ina precise location on the slide or membrane. Companies such as CartesianTechnologies and ProtoGene Laboratories use this technology.

A method for attaching the nucleic acids to a surface is by printing onglass plates, as is described generally by PCT publication WO 95/35505;DeRisi et al., Nature Genetics 14:457-460 (1996); Shalon et al., GenomeRes. 6:639-645 (1996); and Schena et al., Proc. Natl. Acad. Sci. USA 93:10614-10619 (1995). Another method for making microarrays is by makinghigh-density oligonucleotide arrays. Techniques are known for producingarrays containing thousands of oligonucleotides complementary to definedsequences, at defined locations on a surface using photolithographictechniques for synthesis in situ (see, Fodor et al., Science 251:767-773(1991); Pease et al., Proc. Natl. Acad. Sci. USA 91:5022-5026 (1994);Lockhart et al., Nature Biotech 14:1675 (1996); U.S. Pat. Nos.5,578,832; 5,556,752; and 5,510,270, each of which is incorporated byreference).

U.S. Pat. No. 6,110,426 (to Shalon et al.) a method and apparatus forfabricating microarrays of biological samples for large scale screeningassays, such as arrays of DNA samples to be used in DNA hybridizationassays for genetic research and diagnostic applications. U.S. Pat. No.6,221,674 (to Sluka et al.) discloses a process is described forapplying spatially defined reagent areas to a solid phase which ischaracterized in that a liquid containing an adsorptive binding reagentis contacted with spatially defined areas of a solid phase whichcomprises an essentially continuous metal or metal oxide surface for anadequate time period to enable the formation of adsorptive bonds betweenthe binding reagent and the solid phase. A process is described in PCTapplication WO 92/10092 which can be used to generate a plurality ofdifferent structures on a glass support by means of photoreactivecompounds and irradiation using masks. A process is described in U.S.Pat. No. 4,877,745 in which differently functionalized spots can beapplied to plastic supports by means of ink-jet.

Among the vendors of microarrays and microarray technology useage areAffymetrix, Inc. (USA), NimbleGen Systems, Inc. (Madison, Wis., USA),and Incyte Genomics (USA) (producing microarrays for core facilities inlarge industrial and academic departments); Agilent Technologies (USA)and Grafinity Pharmaceutical Design, GmbH (Germany) (which providespecific services such as printing and fingerprinting arrays designedand used by individual researchers); and CLONTECH Laboratories (BectonDickinson Bioscience) and BioRobotics, Ltd. (Great Britain) (whichprovide the basic tools necessary for individual researchers to carryout the entire process of producing microarrays, including printing).See, Gwynne P & Heebner G, “DNA Chips and Microarrays” Science (2001).

In contrast to plastic surfaces, metal and metal oxide surfaces have theadvantage that they can be coated with an exactly defined matrix layerby self-assembly techniques. A self-assembled monolayer (SAM) is formedfor example when organic alkylthiols are adsorbed onto a gold surface,the spontaneous organisation of such a densely packed monolayer beingbased on strong specific interactions between the support material andthe adsorbent. Nuzzo et al., J. Am. Chem. Soc. 105: 4481 (1983). In thismanner it is possible to apply an exactly defined monolayer of a bindingmatrix to the surface of metals such as e.g. gold or silver. Furthermorethe specific binding capability of self-assembled solid phases can befurther optimized by dilution of the specific solid phase reactants asdescribed in EP-A-0 515 615.

The coating of metal surfaces with microstructures based onself-assembled monolayers is also known and can be used to attachcomponents isolated from solid tumor stem cells. Whitesides et al.,Langmuir 10 (1994) 1498-1511 describe a process in which reagents arestamped onto a noble metal surface by means of a special microstructuredsilicone stamp. This enables microstructured monolayers to be generatedwith zones that are spatially separated from one another.Microstructures of self-assembled monolayers on noble metal surfaces canbe formed by irradiation through masks of substrates whose whole area iscovered with thiols and subsequent washing. Hemminger et al., Langmuir10: 626-628 (1994). Spatially separate zones are also formed in thisprocess which are all identically functionalized. A further possibilityof producing reagent spots is firstly to apply gold spots to a supportthat are already spatially separated from one another which are thensubsequently coated with reagents.

The binding of analytes to a functionalized solid phase matrix accordingto the invention can for example be detected by confocal scannerfluorescence microscopy or by plasmon resonance spectroscopy.Ruthenhausler B et al., Nature, 332: 615-617 (1988).

U.S. Pat. No. 6,228,659 describes an apparatus for producing a pluralityof arrays of reagent regions is disclosed. A dispensing assembly in theapparatus has a plurality of heads which are spaced for depositingreagents at selected positions in different array areas in a substrate.

Transcript arrays can be employed for analyzing the transcriptionalstate in a cell, and especially for measuring the transcriptional statesof cells exposed to graded levels of a therapy of interest such asgraded levels of a drug of interest or to graded levels of a diseasestate of interest. In one embodiment, transcript arrays are produced byhybridizing detectably labeled polynucleotides representing the mRNAtranscripts present in a cell (e.g., fluorescently labeled cDNAsynthesized from total cell mRNA) to a microarray. In alternativeembodiments, the cDNA or RNA probe can be synthesized in the absence ofdetectable label and may be labeled subsequently, e.g., by incorporatingbiotinylated dNTPs or rNTP, or some similar means (e.g.,photo-cross-linking a psoralen derivative of biotin to RNAs), followedby addition of labeled streptavidin (e.g., phycoerythrin-conjugatedstreptavidin) or the equivalent. The label for the probe may be selectedfrom the group consisting of biotin, fluorescent, radioactive, andenzymatic labels. When fluorescently-labeled probes are used, manysuitable fluorophores are known, including fluorescein, lissamine,phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5,Cy5.5, Cy7, Fluor X (Amersham) and others (see, e.g., Kricka,Nonisotopic DNA Probe Techniques (Academic Press San Diego, Calif.,1992). It will be appreciated that pairs of fluorophores are chosen thathave distinct emission spectra so that they can be easily distinguished.In another embodiment, a label other than a fluorescent label is used.For example, a radioactive label, or a pair of radioactive labels withdistinct emission spectra, can be used (see Zhao et al, Gene 156:207(1995); Pietu et al., Genome Res. 6:492 (1996); see also, EXAMPLE 21).For example, ³²P can be used.

These methods of attaching transcripts usually attach specificpolynucleotide sequences to very small specific areas of a solidsupport, such as micro-wells of a DNA chip. These hybridization formatsare micro-scale versions of the conventional “reverse dot blot” and“sandwich” hybridization systems. The micro-formatted hybridization canbe used to carry out “sequencing by hybridization” (see, Barinaga,Science 253: 1489 (1991); Bains, Bio/Technology 10: 757-758 (1992).Sequencing by hybridization makes use of all possible n-nucleotideoligomers (n-mers) to identify n-mers in an unknown DNA sample, whichare subsequently aligned by algorithm analysis to produce the DNAsequence (see, U.S. Pat. No. 5,202,231; see also, United Kingdom patentapplication GB 8810400 (1988); Southern et al., Genomics 13: 1008(1992); Fodor et al., Nature 364: 555-556 (1993); Fodor et al., Science251: 767-773 (1991); U.S. Pat. No. 5,143,854.

Probes can be synthesized, in whole or in part, on the surface of asubstrate using a chemical coupling procedure and a piezoelectricprinting apparatus, such as that described in PCT publication WO95/251116 (Baldeschweiler et al.). Alternatively, the probe can besynthesized on a substrate surface using a self-addressable electronicdevice that controls when reagents are added (U.S. Pat. No. 5,605,662).

Furthermore, the probes do not have to be directly bound to thesubstrate, but rather can be bound to the substrate through a linkergroup. The linker groups are typically about 6 to 50 atoms long toprovide exposure to the attached polynucleotide probe. Preferred linkergroups include ethylene glycol oligomers, diamines, diacids and thelike. Reactive groups on the substrate surface react with one of theterminal portions of the linker to bind the linker to the substrate. Theother terminal portion of the linker is then functionalized for bindingthe polynucleotide probe.

Devices and computer systems for forming and using arrays of materialson a chip or substrate are known. For example, PCT International patentapplications WO 92/10588 and WO 95/11995, both incorporated herein byreference, describe techniques for sequencing or sequence checkingnucleic acids and other materials. Arrays for performing theseoperations can be formed in arrays according to the methods of, forexample, the pioneering techniques disclosed in U.S. Pat. Nos.5,445,934, 5,384,261 and 5,571,639, each incorporated herein byreference. Improved methods of forming high-density arrays of peptides,polynucleotides, and other polymer sequences in a short period of timehave been devised using combinatorial solid phase synthesis. Very LargeScale Immobilized Polymer Synthesis (VLSIPS) technology has greatlyadvanced combinatorial solid phase polymer synthesis and paved the wayto clinical application of deoxyribonucleic acid (DNA) array chips suchas those sold under the name GENECHIP™ Kozal et al., Nature Medicine 2:753-759 (1996). VLSIPS technology is disclosed in U.S. Pat. No.5,143,854, PCT International patent applications WO 90/15070), WO92/10092, and WO 95/11995; and Fodor et al., Science 251: 767-777(1991).

Nucleic acid hybridization and wash conditions are chosen so that theprobe “specifically binds” or “specifically hybridizes” to a specificarray site, i.e., the probe hybridizes, duplexes or binds to a sequencearray site with a complementary nucleic acid sequence but does nothybridize to a site with a non-complementary nucleic acid sequence. Asused herein, one polynucleotide sequence is considered complementary toanother when, if the shorter of the polynucleotides is less than orequal to 25 bases, there are no mismatches using standard base-pairingrules or, if the shorter of the polynucleotides is longer than 25 bases,there is no more than a 5% mismatch. Preferably, the polynucleotides areperfectly complementary (no mismatches). It can easily be demonstratedthat specific hybridization conditions result in specific hybridizationby carrying out a hybridization assay including negative controls.Optimal hybridization conditions will depend on the length (e.g.,oligomer versus polynucleotide greater Man 200 bases) and type (e.g.,RNA, DNA, PNA) of labeled probe and immobilized polynucleotide oroligonucleotide. General parameters for specific (i.e., stringent)hybridization conditions for nucleic acids are described in Ausubel etal., Current Protocols in Molecular Biology (Greene Publishing andWiley-Interscience, New York 1987). Useful hybridization conditions arealso provided in, e.g., Tijessen, Hybridization With Nucleic AcidProbes, (Elsevier Science Publishers B.V., 1993) and Kricka, NonisotopicDNA Probe Techniques, (Academic Press, San Diego, Calif., 1992).

When cDNA complementary to the RNA of a cell is made and hybridized to amicroarray under suitable hybridization conditions, the level ofhybridization to the site in the array corresponding to any particulargene will reflect the prevalence in the cell of RNA transcribed fromthat gene. For example, when detectably labeled (e.g., with afluorophore) cDNA complementary to the total cellular mRNA is hybridizedto a microarray, the site on the array corresponding to a gene (i.e.,capable of specifically binding the product of the gene) that is nottranscribed in the cell will have little or no signal (e.g., fluorescentsignal), and a gene for which the encoded mRNA is prevalent will have arelatively strong signal.

U.S. Pat. No. 6,183,968 (to Bandman et al;) discloses polynucleotideprobes that can be used as hybridizable array elements in a microarray,each of the polynucleotide probes having at least a portion of a genewhich encodes a protein associated with cell proliferation or areceptor.

When fluorescently labeled probes are used, the fluorescence emissionsat each site of a transcript array can be, preferably, detected byscanning confocal laser microscopy. In one embodiment, a separate scan,using the appropriate excitation line, is carried out for each of thetwo fluorophores used. Alternatively, a laser can be used that allowssimultaneous specimen illumination at wavelengths specific to the twofluorophores and emissions from the two fluorophores can be analyzedsimultaneously (see Shalon et al., Genome Research 6:639-645 (1996)).Signals are recorded and, preferably, analyzed by computer, usingcommercially available methods. The abundance sort program of theinvention described in U.S. Pat. No. 5,840,484 can be used to tabulateand sort by frequency the mRNA transcripts corresponding to each geneidentified. Since some of the polynucleotide sequences are identifiedsolely based on expression levels, it is not essential to know apriorithe function of a particular gene in solid tumor stem cells.

Transcript image comparisons can be obtained by methods well known tothose skilled in the art. Transcript levels and images can be obtainedand compared, for example, by a differential gene expression assay basedon a quantitative hybridization of arrayed DNA clones (Nguyen et al.Genomics 29:207-216 (1995), based on the serial analysis of geneexpression (SAGE) technology (Velculescu et al. Science 270:484-487(1995)), based on the polymerase chain reaction (Peng et al. Science257:967-971 (1992), based on a differential amplification protocol (U.S.Pat. No. 5,545,522), or based on electronic analysis, such ascomparative gene transcript analysis (U.S. Pat. No. 5,840,484) or theGEMTOOLS gene expression analysis program (Incyte Pharmaceuticals, PaloAlto, Calif., USA). Preferably, comparisons between two or moretranscript profiles are performed electronically.

U.S. Pat. No. 6,215,894 discloses a system for scanning biochip arraysincludes a unique image array identifier recorded for each array, and acomputer-stored record corresponding to each identifier and containingthe parameters of the experiment in the array identified by theidentifier. The system further includes means for accessing a protocollibrary to retrieve the scanning protocols associated with theidentified arrays and then scanning the arrays in accordance with therespective protocols. The resulting image maps generated by the scannersare stored in locations corresponding to the associated identifiers.

Measurement of the translational state may be performed according toseveral methods. For example, whole genome monitoring of protein (i.e.,the “proteome,”) can be carried out by constructing a microarray inwhich binding sites comprise immobilized, preferably monoclonal,antibodies specific to a plurality of protein species encoded by thecell genome.

Use of microarrays. The microarrays describe above can be employed inseveral applications including solid tumor cancer diagnostics,prognostics and treatment regimens, drug discovery and development,toxicological and carcinogenicity studies, forensics, pharmacogenomicsand the like.

In one embodiment, the microarray is used to monitor the progression ofdisease. Physicians can assess and catalog the differences in geneexpression between healthy and cancerous tissues by analyzing changes inpatterns of gene expression compared with solid tumor stem cells fromthe patient. Thus, cancer can be diagnosed at earlier stages before thepatient is symptomatic. The invention can also be used to monitor theefficacy of treatment. For some treatments with known side effects, themicroarray is employed to “fine tune” the treatment regimen. A dosage isestablished that causes a change in genetic expression patternsindicative of successful treatment. Expression patterns associated withundesirable side effects are avoided. This approach may be moresensitive and rapid than waiting for the patient to show inadequateimprovement, or to manifest side effects, before altering the course oftreatment.

Alternatively, animal models which mimic a disease, rather thanpatients, can be used to characterize expression profiles associatedwith a particular disease or condition. This gene expression data may beuseful in diagnosing and monitoring the course of disease in a patient,in determining gene targets for intervention, and in testing noveltreatment regimens.

Also, researchers can use the microarray to rapidly screen large numbersof candidate drug molecules, looking for ones that produce an expressionprofile similar to those of known therapeutic drugs, with theexpectation that molecules with the same expression profile will likelyhave similar therapeutic effects. Thus, the invention provides the meansto determine the molecular mode of action of a drug.

U.S. Pat. Nos. 6,218,122, 6,165,709, and 6,146,830 (au to Friend et al.)discloses methods for identifying targets of a drug in a cell bycomparing (i) the effects of the drug on a wild-type cell, (ii) theeffects on a wild-type cell of modifications to a putative target of thedrug, and (iii) the effects of the drug on a wild-type cell which hashad the putative target modified of the drug. In various embodiments,the effects on the cell can be determined by measuring gene expression,protein abundances, protein activities, or a combination of suchmeasurements. In various embodiments, modifications to a putative targetin the cell can be made by modifications to the genes encoding thetarget, modification to abundances of RNAs encoding the target,modifications to abundances of target proteins, or modifications toactivities of the target proteins. The present invention provides animprovement to these methods of drug discovery by providing thetumorigenic solid tumor stem cells, for a more precise drug discoveryprogram.

An “expression profile” comprises measurement of a plurality of cellularconstituents that indicate aspects of the biological state of a cell.Such measurements may include, e.g., RNA or protein abundances oractivity levels. Aspects of the biological state of a cell of a subject,for example, the transcriptional state, the translational state, or theactivity state, are measured. The collection of these measurements,optionally graphically represented, is called the “diagnostic profile”.Aspects of the biological state of a cell which are similar to thosemeasured in the diagnostic profile, e.g., the transcriptional state, aremeasured in an analogous subject or subjects in response to a knowncorrelated disease state or, if therapeutic efficacy is being monitored,in response to a known, correlated effect of a therapy. The collectionof these measurements, optionally graphically represented, is calledherein the “response profile”. The response profiles are interpolated topredict response profiles for all levels of protein activity within therange of protein activity measured. In cases where therapeutic efficacyis to be monitored, the response profile may be correlated to abeneficial effect, an adverse effect, such as a toxic effect, or to bothbeneficial and adverse effects.

As is commonly appreciated, protein activities result from proteinabundances; protein abundances result from translation of mRNA (balancedagainst protein degradation); and mRNA abundances result fromtranscription of DNA (balanced against mRNA degradation). Therefore,genetic level modifications to a cellular DNA constituent alterstranscribed mRNA abundances, translated protein abundances, andultimately protein activities. RNA level modifications similarly alterRNA abundance and protein abundances and activities. Protein levelmodifications alter protein abundances and activities. Finally, proteinactivity modifications are the most targeted modification methods. As iscommonly appreciated, it is ultimately protein activities (and theactivities of catalytically active RNAs) that cause cellulartransformations and effects. Also, most drugs act by altering proteinactivities.

In one embodiment, cDNAs from two different cells (one being the solidtumor stem cells of the invention) are hybridized to the binding sitesof the microarray. In the case of therapeutic efficacy (e.g., inresponse to drugs) one cell is exposed to a therapy and another cell ofthe same type is not exposed to the therapy. In the case of diseasestates one cell exhibits a particular level of disease state and anothercell of the same type does not exhibit the disease state (or the levelthereof). The cDNA derived from each of the two cell types aredifferently labeled so that they can be distinguished. In oneembodiment, for example, cDNA from a cell treated with a drug (orexposed to a pathway perturbation) is synthesized using afluorescein-labeled dNTP, and cDNA from a second cell, not drug-exposed,is synthesized using a rhodamine-labeled dNTP. When the two cDNAs aremixed and hybridized to the microarray, the relative intensity of signalfrom each cDNA set is determined for each site on the array, and anyrelative difference in abundance of a particular mRNA detected. The useof a two-color fluorescence labeling and detection scheme to definealterations in gene expression has been described, e.g., in Shena etal., Science 270:467-470 (1995). An advantage of using cDNA labeled withtwo different fluorophores is that a direct and internally controlledcomparison of the mRNA levels corresponding to each arrayed gene in twocell states can be made, and variations due to minor differences inexperimental conditions (e.g., hybridization conditions) will not affectsubsequent analyses. Additional guidance is provided in EXAMPLE 21.

U.S. Pat. No. 6,194,158 (to Kroes et al.) for a diagnostic assay forcancer with a DNA chip of specific sequences for measuring expressionlevels of certain sequences within a cancer cell to determine whetherexpression is up- or down-regulated. The DNA chip comprising nucleotidesequences capable of hybridizing to one or more members of a panel ofDNA sequences may be synthesized using commonly available techniques.mRNA is isolated from a normal, non-cancer cell and a cancer cell andhybridized to the DNA chip comprising one of more of the sequences fromthe panel. Hybridization is then detected by any of the availablemethods. In such a manner, sequences that are either overexpressed orunderexpressed in a cancer cell as compared to a normal cell are. In asimilar manner, mRNA from a cancer cell that has been contacted with acompound may be hybridized to sequences on the DNA chip to determinewhether that compound affects expression of a particular sequence. Thepresent invention provides an improvement over this method, in that the“cancer cell” from which mRNA can be isolated is the tumorigenic solidtumor stem cell of the invention.

Gene expression profiles of purified stem cells could give clues for themolecular mechanisms of stem cell behavior. Terskikh A V et al., ProcNatl Acad Sci USA 98(14): 7934-7939 (2001) analyzed hematopoietic stemcells (HSC)-enriched cells by comparison with normal tissue and mouseneurospheres (a population greatly enriched for neural progenitor cells)by comparison with terminally differentiated neural cells, using cDNAmicroarray techniques and in situ hybridization, thus identifyingpotential regulatory gene candidates. The invention provides an improvedmethod of drug discovery over the methods of Terskikh, in that the useof the solid tumor stem cells of the invention can provide a distinctset of drug targets when compared with a patient's normal tissue (suchas from the area of the solid tumor) or compared with the otherpopulations of cells obtained from the solid tumor.

Several other methods for utilizing DNA chips are known, including themethods described in U.S. Pat. Nos. 5,744,305; 5,733,729; 5,710,000;5,631,734; 5,599,695; 5,593,839; 5,578,832; 5,556,752; 5,770,722;5,770,456; 5,753,788; 5,688,648; 5,753,439; 5,744,306 (all of which areincorporated by reference in their entirety). U.S. Pat. No. 5,807,522(to Brown et al.) discloses a method to monitor early changes in a cellthat correlate with levels of a disease state or therapy and whichprecede detectable changes in actual protein function or activity.

Moreover, microarrays of genomic DNA from solid tumor stem cells can beprobed for single nucleotide polymorphisms (SNP), to localize the sitesof genetic mutations that cause cells to become precancerous ortumorigenic. Guidance for such methods are available from the commercialvendors described above and may be found in general genetic methodbooks, such as those described herein.

Vaccines. The solid tumor stem cells of the invention can be used toraise anti-cancer cell antibodies. In one embodiment, the methodinvolves obtaining an enriched population of solid tumor stem cells orisolated solid tumor stem cells; treating the population to prevent cellreplication (for example, by irradiation); and administering the treatedcell to a human or animal subject in an amount effective for inducing animmune response to solid tumor stem cells. For guidance as to aneffective dose of cells to be injected or orally administered; see, U.S.Pat. Nos. 6,218,166, 6,207,147, and 6,156,305, incorporated herein byreference. In another embodiment, the method involves obtaining anenriched population of solid tumor stem cells or isolated solid tumorstem cells; mixing the tumor stem cells in an in vitro culture withimmune effector cells (according to immunological methods known in theart) from a human subject or host animal in which the antibody is to beraised; removing the immune effector cells from the culture; andtransplanting the immune effector cells into a host animal in a dosethat is effective to stimulate an immune response in the animal.

Monoclonal antibodies to solid tumor stem cells may be prepared usingany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (see, e.g., Kozbor, D. et al., J. Immunol.Methods 81:3142 (1985); Cote R J et al. Proc. Natl. Acad. Sci.80:2026-2030 (1983); and Cole S P et al. Mol. Cell. Biol. 62:109-120(1984)).

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used (see, e.g. Morrison S L et al.Proc. Natl. Acad. Sci. 81:6851-6855 (1984); Neuberger M S et al. Nature312:604-608 (1984); and Takeda S et al. Nature 314:452-454 (1985)).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art.

The antibody can also be a humanized antibody. The term “humanizedantibody,” as used herein, refers to antibody molecules in which theamino acid sequence in the non-antigen binding regions has been alteredso that the antibody more closely resembles a human antibody, and stillretains its original binding ability. Antibodies are humanized so thatthey are less immunogenic and therefore persist longer when administeredtherapeutically to a patient.

Human antibodies can be generated using the XenoMouse™ technology fromAbgenix (Fremont, Calif., USA), which enables the generation andselection of high affinity, fully human antibody product candidates toessentially any disease target appropriate for antibody therapy. See,U.S. Pat. Nos. 6,235,883, 6,207,418, 6,162,963, 6,150,584, 6,130,364,6,114,598, 6,091,001, 6,075,181, 5,998,209, 5,985,615, 5,939,598, and5,916,771, each incorporated by reference; Yang X et al., Crit. RevOncol Hemato 38(1): 17-23 (2001); Chadd H E & Chamow S M. Curr OpinBiotechnol 12(2): 188-94 (2001); Green L L, Journal of ImmunologicalMethods 231 11-23 (1999); Yang X-D et al., Cancer Research 59(6):1236-1243 (1999); and Jakobovits A, Advanced Drug Delivery Reviews 31:33-42 (1998). Antibodies with fully human protein sequences aregenerated using genetically engineered strains of mice in which mouseantibody gene expression is suppressed and functionally replaced withhuman antibody gene expression, while leaving intact the rest of themouse immune system.

Moreover, the generation of antibodies directed against markers presentin or on the solid tumor stem cells of the invention can be used as amethod of identifying targets for drug development. The antibodies thatare raised in an immune response to the solid tumor stem cells can beused to identify antigenic proteins on the solid tumor stem cells usingmethods known in the art (Harlow, Using Antibodies: A Laboratory Manual(Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1999)) and canfurther be used to identify polynucleotides coding for such proteins(Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1989); Current Protocols inMolecular Biology, Ausubel et al., eds., (Wiley Interscience, New York,1993)). Once identified, the proteins and polynucleotides can becompared with other proteins and polynucleotides previously identifiedto be involved in cancer. In one embodiment, the XenoMouse™ technologyto produce fully human antibodies can be used to generate antibodiesdirected against drug development targets (see, Jeffrey Krasner, BostonGlobe (Jul. 25, 2001) at F4). The present invention provides animprovement to these antibody-based methods of drug discovery byproviding the tumorigenic solid tumor stem cells, to which the immuneresponse is raised, for a more precise drug discovery program.

The Microarray Network. In addition to the cDNA libraries and DNA arraytechniques described above for systematically studying gene expressionpatterns, one of skill in the molecular biological art can obtainguidance for the use of microarray technology from the University ofMichigan Microarray Network (see also, EXAMPLE 9). Furthermore, theUniversity of Michigan has made a large commitment to bioinformatics, byproviding funding specifically for microarray data management andanalysis, as well as by founding a new Bioinformatics Center for theUniversity.

Alternatively, methods for raising an immune response can take advantageof the “stem cell” qualities of the solid tumor stem cell of theinvention. Solid tumor stem cells, solid tumor stem cell proteinextracts, purified proteins from solid tumor stem cells, or proteinsderived from the expression of cDNAs from solid tumor stem cells (see,above for genetic modification of solid tumor stem cells) to induce animmune response in an animal. The immune response can be directedagainst cancer cells, as shown by standard immunological methods. Forexample, the solid tumor stem cells (enriched populations of isolatedcells) or proteins can be contacted with o dendritic cells in culture,antigen presenting cells in culture, or antigen presenting cells and Tcells in culture. Then antigen-stimulated cells are infused back intothe patient.

Alternatively, the solid tumor stem cells of the invention can begenetically engineered to promote an immune response against the tumorstem cells. For example, hematopoietic stem cells can be engineered tocontain a T-cell receptor targeting a tumor stem cell antigen. See, U.S.Pat. Nos. 5,914,108 and 5,928,638 incorporated by reference. Thus T cellreceptors that recognize antigens expressed by tumor stem cells can beidentified, then cloned into hematopoietic stem cells. The engineeredhematopoietic stem cells can then be transplanted into a patient andallowed to engraft, giving rise to large numbers of T cells that expressreceptors recognizing the tumor stem cells. By increasing the numbers oftumor stem cell-specific T cells the anti-tumor immune response can bepotentiated.

Other means are also available for increasing the anti-tumor immuneresponse, including using the tumor stem cells as the basis of avaccine, using the tumor stem cells to stimulate antigen presentingdendritic cells, and using the tumor stem cells as an innoculum togenerate anti-tumor antibodies. Tumor stem cells can be used as avaccine by killing a patient's tumor stem cells, such as by irradiation,and readministering the killed stem cells back into the patient in aphysiological and immunologically acceptable carrier, for the purpose ofgenerating an immune response against the tumor stem cells. See, U.S.Pat. No. 4,960,716, in which antibodies were raised to membrane vesiclepreparations of breast carcinoma cell cells; U.S. Pat. No. 4,584,268, inwhich anti-human mammary epithelial antibody was produced a membranefraction of delipidated human milk fat globules; both incorporated byreference.

Dendritic cells from a patient can be cultured in vitro and killed tumorstem cells from the same patient can be added to the cultures tostimulate the dendritic cells. The activated dendritic cells, presentingtumor stem cell antigens, can then be re-administered to the patient tostimulate the patient's anti-tumor response. Finally, tumor stem cellscan be administered to an animal such as a mouse, rat, hamster, goat,sheep, rabbit, or donkey to generate antibodies against the tumor stemcells. Preferably, monoclonal anti-tumor stem cell antibodies are madein mouse, rat, or hamster. Monoclonal antibodies that are made in thisway can then be administered to patients, or first humanized (asdescribed above) and then administered to patients, to promote an immuneresponse against the tumor stem cells in the patient.

Furthermore, adenoviral vectors have proven especially useful for genetransfer into eukaryotic cells for vaccine development. Graham F L &Prevec L, In Vaccines: New Approaches to Immunological Problems, Ellis RV ed., 363-390 (Butterworth-Heinemann, Boston, 1992).

Probe for scanning microarrays. The complete sequencing of the humangenome makes possible the identification of the genes expressed by aparticular population of cells.

Probes from enriched populations of tumor stem cells can be made usingmethods known to the art (see, Wang et al., Nature Biotechnology 18: 457(2000). Analysis of gene expression patterns and protein expressionpatterns for tumor stem cells and tumor cell progeny populations can bepreformed by comparing results to known Gene Pattern Databases or toknown Protein Pattern Databases (for example, PROSITE, PRINTS: ProteinMotif Fingerprint Database, BLOCKS, PFAM, DOMO, PRODOM, or otherdatabases). Searches can be preformed, for example, using the BCM SearchLauncher: General Protein Sequence/Pattern Searches (Baylor College ofMedicine, Human Genome Sequencing Center, One Baylor Plaza, Houston,Tex.). Commercially available programs for gene and protein analysis arealso available (such as CGC (from Genetics Computer Group, Inc.) and DNASTRIDER).

The factors involved in the proliferation, differentiation, or survivalof tumor stem cells and tumor stem cell progeny, or their responses tobiological agents can be isolated either by constructing cDNA forlibraries or to probe microarrays from tumor stem cells, or tumor stemcell, non-tumorigenic cancer cells, or normal tumor cells usingtechniques known in the art. cDNA can also be made from any of thedifferent populations after exposure to biological agents or drugs todetermine the response to such manipulations. The libraries from cellsof one population are compared with those of cells of differentpopulations to determine the sequence of gene expression duringdevelopment and to reveal the effects of various biological agents or toreveal new biological agents that alter gene expression in cancer cells.When the libraries are prepared from neoplastic tissue, genetic factorsmay be identified that play a role in the cause of cancer cell growth,for example, by comparing the libraries from the cancerous tissue withthose from normal tissue. This information can be used in the design ofanti-cancer therapies. Additionally, probes can be identified for use inthe diagnosis of various cancers or for use in identifying cells at aparticular stage in tumor development.

Diagnostic and prognostic evaluation of tumors. A variety of methods canbe employed for the diagnostic and prognostic evaluation of tumor andmetastasis, and for the identification of subjects having apredisposition to such conditions. Among the methods well known in theart are the use of bone scans, X-ray imaging, MRI tests, CAT scans, andblood tests for tumor associated antigens (see, American Cancer Society,Cancer Facts and figures 1999: Selected Cancers (1999); American CancerSociety, Breast Cancer Guidelines and Statistics (1999); Kopans BreastImaging. 2^(nd) Edition (J B Lippincott, Philadelphia, Pa., 1998);Potter & Partin, NCCN Practice Guidelines for Early Detection ofProstate Cancer 13(11A) Oncology (November 1999). For additional methodsof detection, see Franklin et al, Breast Cancer Research & Treatment41(1): 1-13 (1996); Kufe et al., Cancer Research 43(2): 851-7 (1983).For bone scans, nuclear medicine imaging can be used. Nuclear medicinemay be used in addition to mammography to help identify certainabnormalities. Nuclear medicine is also a good tool for evaluating themetastasis of cancer into the lymphatic system, other organs andskeletal system. Tumor associated antigens include for example, BCA 225(U.S. Pat. No. 5,681,860); Bladder Tumor Associated Antigen (BTA stattest, ARUP Laboratories, Salt Lake City, Utah); tumor-associated antigenCA125 (Wagner et al., Hybridoma 16(1): 3340 (1997)); 22-1-1 Ag, YH 206,GA 733, CA 125, carcinoembryonic antigen, and sialyl Le^(X) (Sonoda etal., Cancer 77(8): 1501-1509 (1996)). For breast cancer prognosis,cancerous cells can be looked for in the patient's bone marrow. See, forexample, Bruan et al., New Engl. J. Med. (Feb. 24, 2000)).

Such methods may, for example, utilize reagents such as VEGF nucleotidesequences and VEGF antibodies. Specifically, such reagents may be used,for example, for: (1) the detection of the presence or over-expressionof VEGF mRNA relative to the non-carcinogenic tissue state; (2) thedetection of an over-abundance of VEGF protein relative to thenon-carcinogenic tissue state; (3) the detection of hypoxic conditionsin the tumor mass; (4) the detection of the expression of VEGF tyrosinekinase receptors and other angiogenic receptors in adjacent endothelialtissues; and (5) the detection of the expression of oncogenes. Themethods may be performed, for example, by utilizing pre-packageddiagnostic kits comprising at least one specific VEGF nucleotidesequence or VEGF antibody reagent described herein, which may beconveniently used, e.g., in clinical settings, to diagnose patients atrisk for tumor angiogenesis and metastasis.

Further, the expression of different oncogene alleles may be assessedusing these methods. The additional information obtained regarding theexpression of other markers provides guidance for the design ofappropriate therapies to inhibit angiogenesis or tumor proliferationtailored to the molecular stage of the cancer in a particular patient.

Drug discovery. The invention provides a method for identifying a testcompound for reducing solid tumors. The practice of the method can befurther determined using the guidance provided in the EXAMPLES below.The steps of the method include assaying the response of tumor cells tobiological agent and determining the effects of the biological agent onthe tumor stem cell. In other words, the invention provides improvedmethods of drug discovery by the use of the solid tumor stem cells ofthe invention.

Proof of principle of the use of the invention for drug discovery isprovided in drug discovery in EXAMPLE 11 and FIG. 11, where theepidermal growth factor (EGF) receptor (EGF-R) and HER2/neu markers(known to be involved in cancers) were identified on solid tumor stemcells. Accordingly, therapies directed against the EGF-R (e.g., Yang Xet al., Crit. Rev Oncol Hematol. 38(1): 17-23 (2001)) and HER2/neumarkers (see, Breast Disease, Vol. 11, HER2: Basic Research, Prognosisand Therapy, Y. Yarden, ed. (10S Press, Amsterdam, 2000)) can beeffectively targeted to solid tumor stem cells.

The identification of biological pathways is an important part of moderndrug discovery process. Biological pathways in solid tumor stem cellsand other cell populations obtained from solid tumors, particularlypathways involved in drug actions, i.e., pathways that originate at adrug target (e.g., proteins), can be identified for use as shown by U.S.Pat. No. 5,965,352, which is incorporated herein by reference.

In one set of methods, drugs are screened to determine the binding oftest compounds to receptors, in which the binding activates a cell'sbiological pathway to cause expression of reporter polypeptides.Frequently the reporter polypeptides are coded for on recombinantpolypeptides, in which the coding polynucleotide is in operable linkagewith a promoter.

The term is “operably associated” or “operably linked,” as used herein,refer to functionally related polynucleotides. A promoter is operablyassociated or operably linked with a coding sequence if the promotercontrols the translation of the encoded polypeptide. While operablyassociated or operably linked nucleic acid sequences can be contiguousand in the same reading frame, certain genetic elements, e.g., repressorgenes, are not contiguously linked to the sequence encoding thepolypeptide but still bind to operator sequences that control expressionof the polypeptide.

The detectable signal can be fluorescence, absorbence, or luminescence,depending on the reporter polypeptide, which can be, for example,luciferase (firefly luciferase, Vibrio fisceri luciferase, orXenorhabdus luminescens luciferase), green fluorescent protein, greenfluorescent protein variant, chloramphenicol acetyltransferase,β-glucuronidase, β-galactosidase, neomycin phosphotransferase, guaninexanthine phosphoribosyltransferase, thyridine kinase, β-lactamase,alkaline phosphatase, invertase, amylase (for yeast based assays) humangrowth hormone (for activity based assays). The fluorescent detectablesignal can be fluorescence resonance energy transfer (FRET),bioluminescence resonance energy transfer (BRET), time-resolvedfluorescence (TRF) or fluorescence polarization (FP). Where appropriate,the detectable signal is detected by a machine such as a fluorometer,luminometer, fluorescence microplate reader, dual-monochromatormicroplate spectrofluorometer, spectrophotometer, confocal microscope(laser scanner), or a charge-coupled device (CCD). The detectable signalis determined by comparing the amount of signal produced when thereporter polypeptide is expressed in the tumor stem cell with the signalproduced when the reporter polypeptide is not expressed in the tumorstem cell.

Another technique for drug screening provides for high throughputscreening of compounds (see, e.g., PCT application WO 84/03564.) In thismethod, large numbers of different small test compounds are synthesizedon a solid substrate, such as plastic pins or some other surface. Thetest compounds are reacted with solid tumor stem cells, or portionsthereof, and washed. Bound solid tumor stem cells are then detected bymethods well known in the art, using commercially available machineryand methods, for example, the Automated Assay Optimization (AAO)software platforms (Beckman, USA) that interface with liquid handlers toenable direct statistical analysis that optimizes the assays; modularsystems from CRS Robotics Corp. Burlington, Ontario), liquid handlingsystems, readers, and incubators, from various companies using POLARA™(CRS), an open architecture laboratory automation software for a UltraHigh Throughput Screening System; 3P (Plug&Play Peripherals) technology,which is designed to allow the user to reconfigure the automationplatform by plugging in new instruments (ROBOCON, Vienna, Austria); theAllegro™ system or Staccato™ workstation (Zymark), which enables a widerange of discovery applications, including HTS, ultra HTS, andhigh-speed plate preparation; MICROLAB Vector software (Hamilton Co.,Reno, Nev., USA) for laboratory automation programming and integration;and others.

For any of these machines and methods, the assays measure a response thetarget cells (solid tumor stem cells or genetically modified solid tumorstem cells) that provides detectable evidence that the test compound maybe efficacious. The detectable signal is compared to control cells andthe detectable signal identified by subtraction analysis. The relativeabundance of the differences between the “targeted” and “untargeted”aliquots are simultaneously compared using a “subtraction” analysis(differential analysis) technique such as differential display,representational difference analysis (RDA), GEM-Gene ExpressionMicroarrays (U.S. Pat. No. 5,545,531), suppressive subtractionhybridization (SSH) and direct sequencing (PCT patent application WO96/17957). The subtraction analysis can include the methods ofdifferential display, representational differential analysis (RDA),suppressive subtraction hybridization (SSH), serial analysis of geneexpression (SAGE), gene expression microarray (GEM), nucleic acid chiptechnology, or direct sequencing.

The solid tumor stem cell of the invention is particularly useful in thedrug development process because solid tumor stem cells provide alimited and enriched set of targets for drug development. One of themost important steps in rational drug design is the identification of atarget, the molecule with which the drug itself interacts. Frequently,the target will be a receptor on or in a tumorigenic solid tumor stemcell.

Likewise, the genetically modified solid tumor stem cell of theinvention is particularly useful in the drug development. For example,the genetically modified stem cell can contain polynucleotide with apromoter operably linked to the polynucleotide encoding a reporterpolypeptide. The reporter polypeptide is expressed in the tumor stemcell after a receptor of the tumor stem cell is activated by binding toa test compound or inactivated by binding to a test compound. Such adetectable signal makes the genetically modified solid tumor stem cellappropriate for use in high throughput screening (HTS).

The detectable signal can be a result of a positive selection or anegative selection. The positive selection includes manipulations thattest the ability of cells to survive under specific culture conditions,ability to express a specific factor, changes in cell structure, ordifferential gene expression. The selection can be based on the abilityof the solid tumor stem cells or genetically modified solid tumor stemcells to:

(a) Grow or survive under specific culture conditions, such as in vitrocell culture.

(b) Express a specific factor that can be measured, the measurementadaptable for a selection. This factor can be anything that isaccessible to measurement, including but not limited to, secretedmolecules, cell surface molecules, soluble and insoluble molecules,binding activities, activities that induce activities on other cells orinduce other organic or inorganic chemical reactions.

(c) Changes in cell structure, including morphological changes that aremeasured by physical methods such as differential sedimentation,differential light scattering, differential buoyant density,differential cell volume selected by sieving.

(d) Differences in gene expression that can be directly measured,including changes in cell surface markers, changes in biochemicalactivities, any changes that would be re-selected in changes in bindingof fluorescent labeled probes that could be used in conjunction with aFluorescence Activated Cell Sorter (FACS) or any property that can beused as a basis for a selection. Genetically modified solid tumor stemcells containing polynucleotides that express reporter polypeptides areparticularly useful here.

(e) Differences in gene expression that can be indirectly measured,including changes in transcription factor activity that are measured bya synthetic gene construct encoding a selective marker (such as a drugresistance marker or a cell surface marker that could be used in a FACSselection). This category would also include changes in mRNA stability,mRNA localization, mRNA translation control. All of these changes couldbe the basis of a selection because a synthetic construct which iscontrolled by one of these regulatory events could be constructed whichwould drive the expression of an easily selected gene product.

Pharmacogenomics. The invention provides an improved method ofascertaining propensity for malignancy, monitoring the progress ofchemotherapy or other anticancer therapy, screening for re-occurrence ofcancer, or other similar detection of present or potential cancer, wheresuch method detects for the expression of at least one gene which isover- or under-expressed in a potential cancer cell, as compared witheither a solid tumor stem cell isolated from the patient or a collectionof solid tumor stem cells. In one embodiment, the method is the assayingof a biological sample (such as from the patient) to be tested for asignal indicating the transcription of a significant (by comparison withthe solid tumor stem cell) polynucleotide transcript. In addition,screening assays of biological samples are contemplated, where suchassays are conducted during the course of chemotherapy alone, or aftersurgical intervention to treat cancer, to monitor for the continuedpresence or return of cancerous cells.

Other embodiments of the invention. The invention provides an article ofmanufacture (a system or a kit), comprising packaging material and aprimary reagent contained within said packaging material. The primaryreagent is solid tumor stem cell preparation as described above. Thepackaging material includes a label that indicates that the primaryreagent can be used for identifying an agent for reducing solid tumors.

Also, the invention provides a kit for determining the activity level ofa particular polynucleotide or protein in a cell. Such kits containarrays or microarrays containing a solid phase, e.g., a surface, towhich are bound, either directly or indirectly, solid tumor stem cells(enriched populations of or isolated), polynucleotides extracted fromsuch solid tumor stem cells, or proteins extracted from such solid tumorstem cells. The kit may also contain probes that are hybridized or boundto the solid tumor stem cell components at a known location of the solidphase. These probes consist of nucleic acids of known, differentsequence, with each nucleic acid being capable of hybridizing to an RNAspecies or to a cDNA species derived therefrom. In particular, theprobes contained in the kits of this invention are nucleic acids capableof hybridizing specifically to nucleic acid sequences derived from RNAspecies which are known to increase or decrease in response toperturbations correlated to the particular diseases or therapies to bemonitored by the kit. The probes contained in the kits of this inventionpreferably substantially exclude nucleic acids which hybridize to RNAspecies that are not increased or decreased in response to perturbationscorrelated to the particular levels of disease states or therapeuticeffects to be determined by the kit.

The details of one or more embodiments of the invention have been setforth in the accompanying description above. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Other features, objects, and advantagesof the invention will be apparent from the description and from theclaims.

In the specification and the appended claims, the singular forms includeplural referents. Unless defined otherwise in this specification, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. All patents and publications cited in thisspecification are incorporated by reference.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Isolation of Breast Cancer Stem Cells

The purpose of this EXAMPLE is to show structural and cell functionalcharacterization of breast cancer stem cells.

We have developed both a tissue culture and a mouse model to identifythe breast tumor clonogenic cell. In the mouse model, NOD/SCID miceLapidot et al., Nature 367(6464): 645-8 (1994)) are treated with VP-16(Etoposide) (available from commercial sources, such as MoravekBiochemicals, Brea, Calif., USA), and implanted with primary humanbreast cancer tissue (obtained from mastectomy or lumpectomy specimens).Three of five primary tumors formed tumors in this system.

The tumor cells isolated from malignant pleural effusions obtained fromtwo patients (see, Zhang et al., Invasion & Metastasis 11(4): 204-15(1991)) were suspended in Matrigel™ (available from Becton Dickinson,Franklin Lakes, N.J., USA), then were injected into mice. Tumors formedin the injected mice.

By this method, we can generate enough tumor cells for analysis by FACS.We also can generate enough tumor cells to perform biological assays tocharacterize the cells. (For clonogenic assay for detecting rare tumorcells in hematopoietic samples, see U.S. Pat. No. 5,674,694,incorporated by reference).

Phenotypically distinct subsets of tumor cells can be isolated by anysuitable means known in the art, including FACS using a fluorochromeconjugated marker-binding reagent. Any other suitable method includingattachment to and disattachment from solid phase, is also within thescope of the invention. Procedures for separation may include magneticseparation, using antibody-coated magnetic beads, afinity chromatographyand “panning” with antibody attached to a solid matrix, e.g. plate, orother convenient technique. Techniques providing accurate separationinclude fluorescence activated cell sorters, which can have varyingdegrees of sophistication, such as multiple color channels, low angleand obtuse light scattering detecting channels, impedance channels, etc.Dead cells may be eliminated by selection with dyes that bind dead cells(such as propidium iodide (PI), or 7-AAD). Any technique may be employedthat is not unduly detrimental to the viability of the selected cells.

The marker-binding reagent can be directly or indirectly conjugated to amagnetic reagent, such as a superparamagnetic microparticle(microparticle). Direct conjugation to a magnetic particle is achievedby use of various chemical linking groups, as known in the art. Antibodycan be coupled to the microparticles through side chain amino orsulfhydryl groups and heterofunctional cross-linking reagents. A largenumber of heterofunctional compounds are available for linking toentities. A preferred linking group is 3-(2-pyridyldithio)propionic acidN-hydroxysuccinimide ester (SPDP) or4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC) with a reactive sulfhydryl group on the antibody and areactive amino group on the magnetic particle. Alternatively, themarker-binding reagent is indirectly coupled to the magnetic particles.The marker-binding reagent is directly conjugated to a hapten, andhapten-specific, second stage antibodies are conjugated to theparticles. Suitable haptens include digoxin, digoxigenin, FITC,dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods for conjugationof the hapten to a protein, i.e. are known in the art, and kits for suchconjugations are commercially available. Fluorochrome labeled antibodiesare useful for FACS separation, magnetic particles for immunomagneticselection, particularly high gradient magnetic selection (HGMS), etc.Exemplary magnetic separation devices are described in PCT patentapplications WO 90/07380 and WO 96/09550, and European patent 0 438 520.

We have extensively studied the tumors formed by one of the primarytumors and one of the malignant pleural effusion cells. We haveidentified low serum tissue culture conditions in which primary breastcancer cells and cells isolated from a mouse xenograft proliferates forat least 1-3 weeks in tissue culture. Using the in vitro tissue culturemodel, we found that stimulation of a specific receptor can affect thegrowth and survival of breast cancer cells.

We have used the in vitro (tissue culture) and in vivo (mouse xenograft)models of human breast cancer. A human tumor growing in the mouse modelwas harvested and made into a single cell suspension and analyzed byFACS. We found a heterogeneity of expression of cell surface markers ontumor cells. Initially, breast cancer cells isolated from a malignantpleural effusion were separated into groups based upon CD44 expression.Cells were analyzed for expression of markers 520C9 and CD44 (see, FIG.2). 520C9 is known to recognize c-erbB-2 (HER-2/neu). Ring et al.,Molecular Immunology 28:915 (1991). See also, U.S. Pat. No. 4,753,894,which discloses murine monoclonal antibodies that bind selectively tohuman breast cancer cells. Four populations of cells were identifiable.There was a small population of cells that expressed both markers 520C9and CD44, a population that expressed either marker alone, as well as apopulation that expressed neither marker. Cells were isolated withregard to CD44 expression (FIG. 2). CD44⁺ or CD44⁻ cells were tested fortheir ability to proliferate. Marker CD44⁺ tumor cells, but not markerCD44⁻ tumor cells, were able to form colonies in vitro and tumors invivo

TABLE 1 ISOLATED POPULATIONS OF BREAST CANCER STEM CELLS CD44⁺ CD44⁻Colonies in vitro + − Tumorigenicity in mice + − Human breast cancercells were collected using FACS. Analysis of in vitro colonies was donein 2 separate wells using 5,000 cells of the respective phenotype. Invivo growth of sorted cells wa done by injecting mice with 2 × 10⁶marker CD44⁺ or CD44⁻ cells. Mice were analyzed at week in experiment 1and week 4 in experiment 2. The injections of marker CD44⁺ cells, butnot marker CD44⁻ cells, resulted in tumor # formation and growth invitro. The in vitro experiments have been replicated using frozen cellsisolated from the patient and support the in vitro experiments. The invivo experiments have been replicated twice.

These results serve as a proof-of-principle of the stem cell model ofsolid cancer and demonstrate the following:

(a) solid tumor cells are phenotypically and functionally heterogeneous;some tumor cells are tumorigenic, while others have limitedproliferative potential and do not form tumors upon transplantation;

(b) by separating cells by FACS, one can enrich for tumorigenic cells;and

(c) by studying the tumorigenic fractions one can isolate tumor stemcells and more carefully focus strategies for identifying therapeutictargets.

This EXAMPLE shows that that the clonogenic breast cancer tumor cellfrom two tumors express CD44. Other markers also allow the furtherpurification of the breast cancer stem cell. We have analyzed the tumorcells for expression of several antigens. Some antigens withheterogeneous expression patterns include MUC1, Notch-4, annexin V,317G5, CD9, CD24, 260F9, P-glycoprotein and CD49F. 260F9 binds to a 55kilodalton glycoprotein (mucin) B cell surface antigen. Weiner L M etal., Cancer Res. 49:4062-4067 (1989); Gregg, E O. et al J. Immunol,138:4502-4508 (1987). See also, U.S. Pat. No. 4,753,894, which disclosesmurine monoclonal antibodies that bind selectively to human breastcancer cells. Combinations of these markers with CD44 permit increasedenrichment of the tumor stem cells beyond what was achieved with CD44alone.

Remarkably, all of the CD44⁺ (tumorigenic) cells are also B38.1⁺. See,Kufe et al., Cancer Research 43(2): 851-7 (1983) for description ofB38.1 antibody. Annexin, Notch-4, and CD24 expression is heterogeneousby the B38.1⁺ cells. We can thus further purify the breast cancer stemcell from two tumors by analyzing various subpopulations of B381⁺ orCD44⁺ cells. Indeed, we have isolated B38.1⁺CD24⁺, and B38.1⁺CD24⁻ cellsobtained from a primary biopsy and placed them in tissue culture. Onlythe B38.1⁺CD24⁻ cells formed colonies. In another tumor, we isolated the260F9⁺CD24⁻, 260F9⁺CD24^(hi), and the 260F9⁺CD24^(lo) populations ofcells. Only the CD24^(−/lo) population of cells formed tumors (TABLE 2).Note that there is a 5-6 fold enrichment of tumorigenic cells usingB38.1 or 260F9 and CD24.

TABLE 2 ANALYSIS OF TUMORIGENICITY OF CD24⁺ AND CD24^(−/lo) CELLS Tumorformation CD24⁺ CD24^(−/lo) Tumor T1 − + Tumor T2 − + NOD/SCID mice wereinjected with either 50,000-200,000 CD24⁺ or CD24^(−/lo) cells andanalyzed for tumor formation two weeks later.

Example 2 Role for Notch in Breast Cell Proliferation

The purpose of this EXAMPLE is to provide preliminary evidence that inat least two different tumors, Notch 4 is expressed by a minority of thetumorigenic cells. Cells from tumor T1 and tumor T2 from EXAMPLE 1 wereanalyzed for expression of Notch 4. Cells were stained with ananti-Notch 4 polyclonal antibody and analyzed by FACS. 5-15% of cellsexpressed detectable levels of Notch 4. Furthermore, differentpopulations of non-tumorigenic cells express different Notch ligands andmembers of the Fringe family. To determine which Notch RNAs areexpressed by normal breast tissue and breast tumor tissue, we performedRT-PCR using primers specific for each Notch mRNA. Interestingly, Notch1, Notch 3 and Notch 4, but not Notch 2, were expressed by both normalbreast cells and breast tumor cells. We prepared RNA from 100,000 cells.RT-PCR of RNA from normal breast cells or breast tumor cells wasperformed using primers specific for Notch 1, Notch 2, Notch 3, andNotch 4. A PCR product of the predicted size was present for Notch 1, 3,and 4, showing the presence of these markers in these cells. The signalwas lost if the RNA was pretreated with Rnase.

To determine the role of Notch in proliferation, a Notch recombinantligand or agonist peptides of the ligand were added to the medium incultures of normal mammary epithelial cells. Both Notch agonistsstimulated the survival/proliferation of single cells: in the presenceof the Notch agonists, 2-3 times more colonies formed and they includeda higher percentage of large, mixed colonies (40% versus 20%). Whensingle cells were plated at clonal density, we obtained two kinds ofcolonies. There were large colonies made up of hundreds to thousands ofcells, presumably arising from solid tumor stem cells, that were of amixture of myoepithelial cells and ductal epithelial cells. There werealso smaller colonies of cells that appeared to contain only a singlelineage and that probably represented the progeny of restrictedprogenitors.

From a normal breast epithelial cell grown in vitro, bilineage colonies(containing both myoepithelial and ductal epithelial cells) weregenerated by single cells. Myoepithelial cells were identified bystaining with an anti-CALLA antibody. Ductal epithelial cells wereidentified by staining with an anti-ESA antibody. Organoids grown inMatrigel™ in the presence or absence of the Notch agonist peptidebranched and proliferated more in the presence of the Notch agonistpeptide. This agonist peptide also inhibited differentiation, asindicated by the inhibition of casein production. These resultsdemonstrate that these assays also provide a means for detectingmultipotent progenitors from normal breast epithelium. This assay may beused for the purification of normal breast stem cells. These resultsalso demonstrate that Notch has a function in normal breast development.

We then examined Notch 4 expression in breast cancer tumor cells. Highlevels of Notch 4 were expressed on a minority of the tumor cells. Whenthe B38.1 population, which identifies the tumorigenic population, wasanalyzed for Notch 4 expression, a distinct minor population of cellsthat were B38.1^(low), Notch 4⁺ became apparent.

We sorted Notch 4⁺ and Notch 4⁻ cells and analyzed them in vitro.Surprisingly, neither population grew in tissue culture. There were twopossible explanations. First, it was possible that interaction betweenthe two populations of cells was required for cell growth. Next, theantibody may be either an agonist or an antagonist of Notch 4 and mayinhibit tumor cell growth. To distinguish between these possibilities,tumor cells were incubated with the anti-Notch 4 antibody, and thenassayed in vitro. Breast cancer cells were placed in tissue cultureafter exposure to an anti-Notch 4 antibody. Cells were incubated on icefor 1 hr. in HBSS containing no anti-Notch 4 antibody, anti-Notch 4antibody, or anti-Notch 4 antibody that had been preincubated with thepeptide used to generate the antibody. Cells were also grown in thepresence of soluble Delta and without soluble Delta as a control.

While control cells grew in tissue culture and formed colonies, cellsincubated with the Notch 4 antibody did not. When the anti-Notch 4antibody was incubated with the peptide used to generate the antibodybefore addition to the cells, growth was restored. To confirm a role forNotch for growth in vitro, cells were incubated in serum free conditionsin media that contained soluble Delta, a Notch ligand, or a controlculture without Delta. The cultures with Delta formed many colonies,whereas only a few small colonies formed without soluble Delta or incultures using cells exposed to the anti-Notch 4 antibody. When theantibody was preincubated with the peptide, the antibody no longerblocked proliferation. Thus, the Notch 4 pathway is critical for breastcancer cell growth in vitro.

Tumor cells were exposed to the anti-Notch 4 antibody and then the cellswere injected into mice to determine whether the antibody inhibits tumorformation. 35,000 tumor cells were incubated with no antibody, theanti-Notch 4 antibody, or the anti-Notch 4 antibody that had beenpreincubated with the peptide used to generate the antibody. After twoweeks, the diameter of tumors formed by the cells exposed to theanti-Notch 4 antibody was 40% smaller than the control tumors. Thus thecells that were exposed to the anti-Notch 4 antibody formed smallertumors than cells that were exposed to the anti-Notch 4 antibody thatwas incubated with the peptide used to generate the antibody.

Expression of Notch, Notch ligands, and Fringe family members insubpopulations of breast cancer tumor cells. We examined the expressionof members of the Notch receptor family, Notch ligands and Notch signalmodifying proteins in populations of breast tumor cells. We initiallyfocused on the markers in the Notch 4⁺ and Notch 4⁻ populations. Onehundred Notch 4⁺ or Notch 4⁻ cells were isolated by FACS (see, FIG. 4).Forty rounds of PCR were performed to detect the indicated mRNA. TheNotch 4⁺ population expressed Notch 1, Notch 4, and Jagged (a Notchligand). The Notch 4⁻ population expressed Notch 1 and Notch 3 as wellas Jagged. Interestingly, Notch 3 (which may inhibit signaling throughother Notch receptors) was not expressed by the Notch 4⁺ population.

Summary. We have developed in vitro and in vivo assays for normal humanbreast cells and human breast tumor stem cells. In two different tumorsarising in the NOD/SCID mouse model, the tumor cells were heterogeneouswith respect to the expression of several cell surface markers. In bothtumors, the phenotype of the clonogenic tumor stem cell wasB38.1⁺CD44⁺CD24^(−/lo). The same population was found to be theclonogenic in the in vitro assay. This B38.1⁺ population can be furthersubdivided using several additional markers. In vitro and in vivoevidence strongly implicates the Notch pathway, especially Notch 4, asplaying a central pathway in tumorigenesis.

Example 3 Mouse Xenograft Model

We have developed a xenograft model in which we have been able toestablish tumors from primary breast tumors via injection of tumors inthe mammary gland of severely immunodeficient mice. Xenograft tumorshave been established from mastectomy specimens of all five patientsthat have been tested to date. We have also been able to establishtumors from three malignant pleural effusions. NOD/SCID mice weretreated with VP-16, and implanted with primary human breast cancertissue. Tumor cells isolated from three malignant pleural effusionssuspended in Matrigel™ were injected into mice and also formed a tumors.This enabled us to generate enough malignant tumor cells to facilitateanalysis by flow-cytometry and assay for the ability of differentsubsets of cells to form tumors. We have extensively studied the tumorsformed by one of the primary tumors and one of the malignant pleuraleffusion cells. Furthermore, in the three tumors that we have attemptedto do so, we have been able to make single-cell suspensions and transferthe tumors. These improvements in the xenograft assay have allowed us todo biological and molecular tests to characterize the clonogenic breastcancer cell. In addition, we have found tissue culture conditions inwhich primary breast cancer cells and cells isolated from a mousexenograft tumor have proliferated for a short period of time (−1-3weeks) in tissue culture.

A human tumor growing in the mouse model was harvested, made into asingle cell suspension, and analyzed by FACS. There was heterogeneity ofexpression of cell surface markers by tumor cells. Initially, breastcancer cells isolated from a malignant pleural effusion were separatedinto groups based upon CD44 expression. Cells were analyzed forexpression of markers 520C9 and CD44. Three populations of cells wereidentifiable. There was a small population of cells that expressed bothmarkers 520C9 and CD44, a population that expressed either marker alone,as well as a population that expressed neither marker. Cells wereisolated with regard to CD44 expression. CD44⁺ or CD44⁻ cells weretested for their ability to proliferate. Marker CD44⁺ tumor cells, butnot marker CD44⁻ tumor cells, were able to form colonies in vitro andtumors in vivo (TABLE 3). Note that isolation of CD44⁺ cells results ina 2-fold enrichment of the tumorigenic cells.

TABLE 3 HUMAN BREAST CANCER CELLS COLLECTED USING FACS CD44⁺ CD44⁻Colonies in vitro + − Tumorigenicity in mice + − Analysis of in vitrocolonies was done in 2 separate wells using 5,000 cells of therespective phenotype. In vivo growth of sorted cells was done byinjecting mice with 2 × 10⁶ marker CD44⁺ or CD44⁻ cells. Mice wereanalyzed at week 3 in test 1 and week 4 in test 2. The injections ofmarker CD44⁺ cells, but not marker CD44⁻ cells, resulted in tumorformation and growth in vitro. The in vitro tests have been replicatedusing # frozen cells isolated from the patient and support the in vitrotests. The in vivo tests have been replicated twice.

These results show that the clonogenic breast cancer tumor cellexpresses CD44. We have begun to search for other markers that mightallow us to further purify the breast cancer stem cell. To do this, wehave analyzed the tumor cells for expression of several antigens.

Surprisingly, all of the CD44⁺ (tumorigenic) cells were also B38.1⁺.Indeed, we have isolated B38.1⁺CD24⁺ cells and B38.1⁺CD24⁻ cellsobtained from a primary biopsy and placed them in tissue culture. Onlythe B38.1⁺CD24⁻ cells formed colonies.

We next isolated cells from two of the tumors based upon expression ofmarker CD24. In tumor T2, we isolated the CD24⁻, the CD24^(lo) and theCD24^(hi) populations. In both cases, only the CD24^(−/lo) populationsformed tumors (TABLE 4). Note that there was a 5-6-fold enrichment oftumorigenic cells using B38.1 and CD24.

TABLE 4 FRACTIONS OF HUMAN BREAST CANCER CELLS ISOLATED BYFLOW-CYTOMETRY Mouse tumor formation CD24⁺ CD24^(−/lo) Tumor T1 − +Tumor T2 − + Mice were injected with 50,000 CD24⁺ or CD24^(−/lo) cells.The formation of tumors was determined 4 weeks after injection.

Analysis of tumors arising from the CD24⁻ cell population. By the solidtumor stem cell model, CD24⁻ cells give rise to tumors that contain bothCD24⁺ and CD24⁻ cells. To test this hypothesis, secondary transplantswere performed using B38.1⁺CD24⁻ cells.

The resultant tumors were removed and the cells were re-analyzed withrespect to B38.1 and CD24 expression. As predicted by the stem cellmodel, cells obtained from a tumor arising from transplanted B38.1⁺CD24⁻cells were heterogeneous with respect to expression of both B38.1 andCD24. The marker expression pattern of the cells isolated from the tumorinitiated by the B38.1⁺CD24⁻ cells was similar to that of the originaltumor.

These results are a proof-of-principle of the solid tumor stem cellmodel of solid cancer and demonstrate the following:

(1) tumor cells are phenotypically and functionally heterogeneous;

(2) by separating cells by FACS, one can enrich for tumorigenic cells;and

(3) by testing the tumorigenic fractions, one can isolate tumor stemcells and more carefully focus strategies for identifying therapeutictargets.

Example 4 Analysis of Primary Breast Tumor Cells in a Mouse Model

We have established tumors from eight patients in our mouse model. Wealso characterize tumors established from three primary tumors and twopleural effusions. Two are fast growing tumors and three are slowgrowing tumors.

The phenotype of the tumorigenic cell is determined for each differenttumor. For analysis, a tumor is removed from the mice and made into asingle cell suspension. We first confirm the solid tumor stem cell modelof the invention and that the phenotype of tumorigenic cells is indeedB38.1⁺CD44⁺CD24^(−/lo). In all tumors, we do limiting dilution analysisof cells isolated by FACS based upon expression of these markers.

Based on our preliminary data, the antibody cocktail that leads to thegreatest purification of the putative tumorigenic cell follows:anti-38.1-APC, anti-CD44-FITC, and anti-CD24-PE, anti-CD3-cytochrome,anti-CD2-cytochrome, anti-CD10-cytochrome, anti-CD14-cytochrome,anti-CD16-cytochrome, anti-CD31-cytochrome, CD45-cytochrome,CD140b-cytochrome, anti-CD64-cytochrome, anti-ESA-Phar-red and 7AAD (aviability marker). All antibodies labeled with cytochrome are consideredto be part of a lineage cocktail (LINEAGE). FACS is used to isolate theputative breast cancer stem cell, which in xenograft tumor T1 are theCD44⁺CD24⁻/loLINEAGE⁻ population of cells, theCD44⁺B38.1⁺CD24^(−/lo)LINEAGE⁻ population of cells are more enriched,and the ESA⁺CD44⁺B38.1⁺CD24^(−/lo)LINEAGE-⁻ population of cells are mostenriched for the breast cancer stem cell. The different populations ofmalignant cells are tested in the in vivo and in vitro models to confirmthat the phenotype of the primary tumorigenic cell does not change whilegrowing in the mouse xenograft model. As we progressively enrich thetumorigenic cells, the number of cells and time needed to form a tumordecreases.

Example 5 Role for Notch in Breast Cell Proliferation

The Notch protein is a receptor for the growth/survival factors Deltaand Jagged (Panin et al., Nature 387(6636): 908-912 (1997); Perron etal, Cellular & Molecular Life Sciences 57(2): 215-23 (2000); Shimizu etal., Journal of Biological Chemistry 274(46): 32961-9 (1999)). In somenormal stem cells, Notch is known to play a role in proliferation,survival and differentiation. Apelqvist et al., Nature 400(6747): 877-81(1999); Berry et al., Development 124(4): 925-36 (1997); Yasutomo etal., Nature 404(6777): 506-10 (2000); Morrison, Cell 101: 499-510(2000). In certain situations, stimulation of Notch can promote stemcell self-renewal while in other situations it can promotedifferentiation. Delta activates all four Notch receptors.

We examined Notch 4 expression in the breast cancer tumor cells. Notch 4was expressed on the minority of the tumor cells. When the B38.1population, which identifies the tumorigenic population, was analyzedfor Notch 4 expression, a distinct minor population of cells that wereB38.1^(lo) and Notch 4⁺ became apparent.

We sorted Notch 4⁺ and Notch 4⁻ cells and analyzed their ability to formcolonies in vitro. Surprisingly, neither population grew in tissueculture. There are two possible explanations. First, it is possible thatinteraction between the two populations of cells is required for cellgrowth. Alternately, the antibody may be either an agonist or antagonistof Notch 4 and inhibition or activation of the receptor may inhibittumor cell growth.

To distinguish between these possibilities, unseparated tumor cells wereincubated with the anti-Notch 4 antibody, and then assayed for theability to form colonies in vitro. While control cells grew in tissueculture and formed colonies (FIG. 5A), cells incubated with the Notch 4antibody did not grow (FIG. 5B). When the anti-Notch 4 antibody waspre-incubated with the peptide used to generate the antibody (thispeptide should theoretically block binding of the antibody to thecells), the ability of the tumor cells to form colonies was restored(FIG. 8C). To confirm that Notch regulates colony formation by tumorcells, cells were incubated in medium with or without soluble Delta(FIG. 5A and FIG. 5D, respectively). The cultures with Delta formed manycolonies (FIG. 5A), whereas only a few small colonies formed in mediumlacking soluble Delta or in cultures using cells exposed to theanti-Notch 4 antibody (FIG. 5B, FIG. 5D, and FIG. 5E). Taken together,these results show that the Notch 4 pathway regulates theproliferation/survival of breast cancer cells in cells and that theanti-Notch 4 antibody blocks activation in vitro.

Next, tumor cells were incubated with the anti-Notch 4 antibody and thenthe cells were injected into mice to determine whether the antibodyinhibits tumor formation. The cells that were exposed to the anti-Notch4 antibody formed smaller tumors than cells that were exposed to theanti-Notch 4 antibody that was incubated with the peptide used togenerate the antibody before addition to the cells. Approximately350,000 tumor cells were incubated with no antibody, the anti-Notch 4antibody, or the anti-Notch 4 antibody that had been incubated with thepeptide used to generate the antibody. After two weeks, the diameter oftumors formed by the cells exposed to the anti-Notch 4 antibody was 40%smaller than either of the control tumors. The simplest explanation ofthe in vitro and in vivo assays is that Notch 4 activation promotesproliferation/survival of stem cells and that the anti-Notch 4 antibodyblocks receptor activation.

Example 6 Further Characterization of Breast Cancer Stem Cells

Human breast cancer tumors have been established in NOD/SCID mice fromfive tumors (mastectomy specimens of three primary breast tumors and twomalignant pleural effusions from metastatic breast cancer). The fivetumors were initially used to understand neoplastic cell markerheterogeneity. Based upon these results, experiments using cellsisolated from primary tumors directly after removal from a patient weredone to confirm that the mouse xenograft model accurately reflects humanbreast cancer.

CD44 expression and tumorigenicity. Cells obtained from the metastaticbreast cancer, designated T1, and one primary breast tumor, designatedT2, were chosen to expand in the mice to obtain sufficient cells foranalysis. To accomplish this, 10⁶-10⁷ pleural effusion cells from T1 (afrozen sample) or a 1-2 mm³ piece of T2 (fresh biopsy sample) were grownin the mouse mammary fat pad for 1 to 3 months. The resulting humantumors were then harvested, made into a single cell suspension andanalyzed by flow-cytometry for expression of several different antigens.Contaminating mouse cells were gated out of the analysis by eliminatingcells expressing mouse H-2K (major histocompatibility complex class I)and dead cells were eliminated using a viability dye.

As predicted by the solid tumor stem cell model, cells displayedheterogeneous expression of a variety of cell surface-markers includingCD44 and B38.1. To determine whether these markers could distinguishtumorigenic from non-tumorigenic cells, CD44⁺ and CD44⁻ cells (FIG. 6)were isolated from these in vivo passaged T1 or T2 cells and NOD/SCIDmice were injected with CD44⁺ or CD44⁻ cells.

Identification of other informative markers. Between 6 and 12 weeksafter injecting, mice were examined for tumors by observation andpalpation, then all mice were necropsied to look for growths atinjection sites that might be too small to palpate. All of the CD44⁺injections gave rise to visible tumors, but none of the CD44⁻ injectionsformed detectable tumors (TABLE 5). Next, cells from first passage T1and T2 were sorted based upon the expression of B38.1 and injected intomice. Tumors appeared from all injections of B38.1⁺ cells but no tumorformation was detected from B38.1⁻ cells (TABLE 5). Thus, tumorigeniccells from both passaged tumors were B38.1⁺CD44⁺.

TABLE 5 Tumorigenicity of different populations of tumor T1 and tumor T2cells # tumors/# of injections Cells/injection 8 × 10⁵ 5 × 10⁵ 2 × 10⁵T1 cells CD44⁻ 0/2 0/2 — CD44⁺ 2/2 2/2 — B38.1⁻ 0/2 0/2 — B38.1⁺ 2/2 2/2— CD24⁺ — — 1/6 CD24⁻ — — 6/6 T2 cells CD44⁻ 0/2 0/2 — CD44⁺ 2/2 2/2 —B38.1⁻ 0/2 0/2 — B38.1⁺ 2/2 2/2 — CD24⁺ — — 1/6 CD24⁻ — — 6/6 Cells wereisolated by flow cytometry as described in FIG. 2 based upon expressionof the indicated marker and assayed for the ability to form tumors afterinjection of 2-8 × 10⁵ cells into the mammary fat pad of NOD/SCID mice.The number of tumors that formed/the number of injections that wereperformed is indicated for each population of cells.

The tumors were also heterogeneous for CD24 expression. When 200,000CD24⁺ or CD24^(−/lo) cells were injected into NOD/SCID mice, all miceinjected with CD244^(−/lo) cells grew tumors (TABLE 5). Although notumors could be detected by palpation in the locations injected withCD24⁺ cells, two of twelve mice injected with CD24⁺ cells did containsmall growths at the injection site that were detected only uponnecropsy. The sites injected with CD24^(−/lo) cells, by contrast, allhad tumors greater than 1 cm in diameter that were readily apparentvisually and by palpation. Since it is extremely difficult to completelyeliminate CD24⁻ cells from the CD24⁺ fraction by flow-cytometry, thesmall growths likely represent contamination by the 1-3% of CD24⁻ cellsthat are typically present in the sorted CD24⁺ population. Alternately,the small growths might have arisen from CD24⁺ cancer cells that hadreduced proliferative capacity. At present we cannot distinguish betweenthese possibilities. Nonetheless, all of the cells that producedpalpable tumors in this xenograft model were B38.1⁺CD44⁺CD24^(−/lo).

Several antigens associated with normal cell types, to which we refercollectively as LINEAGE (CD2, CD3, CD10, CD14, CD16, CD19, CD31, -CD45,-CD64 and -CD140b) markers, were found not to be expressed by the cancercells based on analyses of tumors that had been passaged multiple timesin mice. By eliminating LINEAGE⁺ cells, normal human leukocytes,endothelial cells, and fibroblasts were eliminated, especially fromunpassaged or first passage tumors.

Flow-cytometric analysis of four of the tumors established in NOD/SCIDmice revealed that all of these tumors were heterogeneous with respectto B38.1, CD44, and CD24 expression (see, for example, FIG. 7A, FIG.7B). However, each of the tumors contained a distinct population ofB38.1⁺CD44⁺CD24^(−/lo) cells. In four independent tumors that wereanalyzed after one passage in NOD/SCID mice, the frequency ofB38.1⁺CD44⁺CD24^(−/lo) cells was 9.5.+−.4.5% (mean±standard deviation)of live human tumor cells. Seven weeks after inoculation, the injectionsites of B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells andB38.1⁺CD44⁺CD24⁺LINEAGE⁻ cells were examined by histology. TheB38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ injection sites contained tumors greaterthan 1 cm in diameter while the B38.1⁺CD44⁺CD24⁺LINEAGE⁻ injection sitescontained no detectable tumors. Only normal mouse mammary tissue wasseen by histology at the sites of the B38.1⁺CD44⁺CD24⁺LINEAGE⁻injections (FIG. 7K), whereas the tumors formed byB38.1⁺CD44⁺CD24⁻/loLINEAGE⁻ cells contained malignant cells as judged bymorphology in hematoxylin and eosin stained sections (FIG. 7L). Evenwhen B38.1⁺CD44⁺ CD24⁺LINEAGE⁻ injection sites were examined after 11weeks, no tumors were detected.

Enrichment of tumorigenic cells using B38.1, CD44, CD24, and LINEAGEmarkers. T1, T2, and T3, a third tumor that had been passaged once inNOD/SCID mice, were tested to determine whether tumorigenic cells couldbe enriched based upon expression of B38.1, CD44, CD24 and LINEAGEmarkers. In all cases, the B38.1⁺CD44⁺CD24^(−/lo) cells were markedlyenriched for tumorigenic cells. When injecting unsorted T1 or T2 cells,5×10⁴ cells consistently gave rise to a tumor, but 10⁴ cells gave riseto tumors in only a minority of cases (TABLE 6). In contrast whenB38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells were isolated from T1 or T2 (FIG.7), as few as 10³ cells from this population were able to give rise totumors in all cases (TABLE 6, FIG. 7J and FIG. 7L). Cells that wereB38.1⁺CD44⁺LINEAGE⁻ but CD24⁺ failed to form tumors. These data indicatethat the B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ population is at least 10-foldenriched for the ability to form tumors in NOD/SCID mice relative tounfractionated tumor cells. This population accounted for 4-17% of thecells in T1 and T2 (5-23% of cancer cells).

Example 7 Analysis of Primary Human Tumor Cells

To rule out the possibility that the tumorigenic activity in theenriched population of EXAMPLE 7 arose as a result of propagation in thexenograft model, we isolated unfrozen pleural effusion breast cancercells based upon expression of CD44 and CD24. Approximately 11% of theneoplastic cells were CD44⁺CD24^(−/lo)LINEAGE⁻ and 75%CD44⁺CD24⁺LINEAGE⁻. All three injections of 100,000CD44⁺CD24^(−/lo)LINEAGE⁻, but none of the CD44⁺CD24⁺LINEAGE⁻ cells,formed tumors (TABLE 6). We next tested whether theB38.1⁺CD44⁺CD24^(−/lo) population from unpassaged breast tumor specimens(FIG. 7I) was tumorigenic. Of four independent tumors that were analyzedafter being passaged in mice, there were only enough frozen unpassagedcells available of T1 to permit flow-cytometry. In addition, frozenunpassaged cells from a fifth patient (T5) were analyzed. Five percentof unpassaged T1 cells and 35% of unpassaged T5 cells wereB38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻.

All 40,000 B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells or 40,000B38.1⁺CD44⁺CD24⁺LINEAGE⁻ cells that were isolated by flow-cytometry fromunpassaged T1 were injected into a single mouse. From the unpassaged T5cells, 60,000-100,000 cells of the B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ (FIG.7I) and B38.1⁺CD44⁺CD24⁺LINEAGE⁻ (FIG. 7F) populations were isolated andinjected into each of three NOD/SCID mice. The CD24^(−/lo) populationformed tumors from the T1 cells and in 2 of 3 injections from T5 cells,but the CD24⁺ population never formed tumors, demonstrating that thetumorigenic population identified in the passaged tumors also existed inthe unpassaged T1 and T5 samples. When tumors that arose from theunpassaged T1 cells were analyzed, the frequency ofB38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells was similar in both passaged andunpassaged T1 samples.

TABLE 6 Tumorigenic breast cancer cells are highly enriched in the CD44⁺B38⁺CD24⁻ population Number of tumors/number of injections for each celldose Cells/injection 5 × 10⁵ 1 × 10^(s) 5 × 10⁴ 2 × 10⁴ 1 × 10⁴ 5 × 10³1 × 10³ 5 × 10² 2 × 10² T1 cells (xenograft) Unsorted 4/4 4/4 6/6 — 2/6— 0/6 — — B38.1⁺CD44⁺CD24⁺ — — — 0/5 0/5 0/5 0/5 — — B38.1⁺CD44⁺CD24⁻ —— — 5/5 5/5 5/5 5/5 — — ESA⁺B38⁺CD24⁻ — — — — — —  8/8* 2/2 1/2ESA⁻B38⁺CD24⁻ — — — — — —  0/8* 0/2 0/2 T2 cells (xenoeraft) Unsorted4/4 4/4 4/4 — 1/6 — 0/6 B38.1⁺CD44⁺CD24⁺ — — — 0/5 0/5 0/5 0/5 — —B38.1⁺CD44⁺CD24⁻ — — — 5/5 5/5 5/5 5/5 — — T3 cells (xenograft)B38.1⁺CD44⁺CD24⁺ — — — — 0/2 — — — — B38.1⁺CD44⁺CD24⁻ — — — — 2/2 — — —— T5 cells (primary cells) CD44⁺CD24⁺ 0/3 CD44⁺CD24' 3/3 T1 cells(primary cells) B38.1⁺CD44⁺CD24⁺ 0/1 B38.1⁺CD44⁺CD24⁻ 1/1 T6 cells(primary cells) B38.1⁺CD44⁺CD24⁺ 0/1 0/2 B38.1⁺CD44⁺CD24⁻ 1/1 1/2 Cellswere isolated from first passage tumor T1, tumor T2, or tumor T3 cells.B38.1⁺CD44⁺CD24^(−/lo)LINEAGE and B38.1⁺CD44⁺CD24⁺LINEAGE⁻ cells wereisolated by flow-cytometry as described in FIG. 3. The indicated numberof cells of each phenotype was injected into the mammary fat pad ofNOD/SCID mice. The number of tumors that formed from injections of eachgroup of cells is indicated. Note that isolation of #B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells results in more than a ten-foldenrichment in tumorigenic cells and that no tumors arose from injectionof B38.1⁺CD44⁺CD24⁺ LINEAGE⁻ cells. When cells were sorted as ESA⁺ inaddition to B38.1⁺CD24⁻, injections of as few as 200 cells gave rise toa tumor, an approximately 50-fold enrichment in tumorigenicity relative# to unsorted cells. *for these injections 2 × 10³ cells were usedinstead of 1 × 10³.

Example 8 Further Characterization of the Cell Populations from a BreastCancer Tumor

In one tumor, tumorigenic cells were further enriched by selecting theESA⁺ subset of the B38.1⁺CD44⁺CD24^(−/lo) population. WhenESA⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells were isolated from T1 (FIG. 8A), asfew as 200 cells from this population were able to give rise to a tumorwhile injection of 2,000 B38.1⁺CD24^(−/lo)LINEAGE⁻ but ESA⁻ cells failedto form tumors in any case (TABLE 6). At least 10,000 unfractionatedcells were required to form any tumors. Thus, theESA⁺1338.1⁺CD24^(−/lo)LINEAGE⁻ population was at least 50 fold enrichedfor the ability to form tumors relative to unsorted tumor cells. TheESA⁺B38.1⁺CD2-4^(−/lo)LINEAGE⁻ population accounted for 2-4% of T1 cells(2.5-5% of cancer cells). The ESA⁺B38.1⁺CD24^(−/lo)LINEAGE⁻ and thenon-tumorigenic cells were examined by cytology and both populationsconsisted of malignant cells that were virtually indistinguishable inappearance.

Example 9 Use of Cancer Stem Cells to Improve Tissue Culture Conditionsfor Growth of Breast Cancer Cells

In tissue culture, it has long been known that only a minority ofprimary tumor cells are capable of forming colonies. According to thesolid tumor stem cell model, while the non-tumorigenic population ofcells might retain some ability to proliferate, the tumorigenicpopulation of cells would have a greater capacity to do so. Equalnumbers of the tumorigenic phenotype and the non-tumorigenic cells,isolated either directly from a primary tumor or from tumor T1 and tumorT4 growing in mice, were seeded at clonal density in tissue cultureplates and colony formation was measured. The primary patient cells werefrom an aliquot of frozen primary cells from a tumor that easily forms abreast cancer cell line and proliferates well in tissue culture.

Cytology showed that each population contained cancer cells ofepithelial origin FIG. 9).

Initially, colonies formed from not only the tumorigenic cells but insome tumors also from the non-tumorigenic cells (FIG. 9). However, byday twelve, the colonies formed by the tumorigenic cells had grown whilethe non-tumorigenic cells had died (FIG. 9). By day 14, onlyB38.1⁺CD44⁺CD24⁺LINEAGE⁻ cells formed numerous large colonies of cancercells (FIG. 9C).

We have passed these cells in tissue culture over 3 passages, and theepithelial neoplastic cells continue to proliferate. By contrast, thenon-tumorigenic cells were capable of forming only small coloniesconsisting of 2-4 cells that stopped proliferating after a few days(FIG. 9C).

These results illustrate two important points. First, the phenotype ofthe stem cells is not an artifact of the immune-deficient mouse assay.The fact that the phenotypically identical population of cells hadincreased proliferative ability in both the mouse xenograft model andthe in vitro assays shows that this population is the tumorigenicpopulation of cells found in patients with breast cancer.

Second, at least some of the non-tumorigenic cells have the capacity forlimited proliferation. This shows that the markers used to identify thenon-tumorigenic population were not simply selecting for cells thatlacked viability.

These results of this EXAMPLE provides a solution to the problem thatthe use of the in vitro clonogenic assay to predict chemotherapy drugsensitivity frequently does not predict a particular patient's responseto therapy. In one of the tumors used in this EXAMPLE, thenon-tumorigenic population of cells formed colonies almost asefficiently as the tumorigenic cells (the solid tumor stem cells of theinvention) on day 4. In using this tumor for a drug-screening assay, ifthe non-tumorigenic and tumorigenic cells that are clonogenic in vitrorespond differently to a particular test compound, then the assay,especially if scored early, would not have reflected the ability of thetest compound to eliminate breast cancer stem cells in vivo.

Example 10 The Stem Cell Population Regenerates Both the Stem Cell andthe Non-Tumorigenic Populations of Cells

According to the deterministic solid tumor stem cell model (FIG. 1B),B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ tumorigenic cells form tumors thatcontain additional B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ tumorigenic cells aswell as phenotypically heterogeneous populations of non-tumorigeniccells. Classical models would suggest that tumors that form fromB38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells consist solely of expanded numbersof B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells.

Tumors arising from 1,000 B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells weredissociated and analyzed by flow-cytometry with respect to B38.1, CD44and CD24 expression (FIG. 10). As expected from the deterministic solidtumor stem cell mode, cells obtained from a tumor arising fromtransplanted B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells were heterogeneouswith respect to expression of all markers (FIG. 10C and FIG. 10F). Inboth cases, the marker expression pattern of the cells isolated from thetumor initiated by the B38.1⁺CD44⁺CD24⁻/loLINEAGE⁻ cells was similar tothat of the original tumors (compare FIG. 10A and FIG. 10B with FIG. 10Cand FIG. none of the B38.1⁺CD44⁺CD24⁺LINEAGE⁻ population, gave rise tonew tumors.

This EXAMPLE demonstrates that the B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻population gives rise to both additional tumorigenicB38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells and phenotypically distinctnon-tumorigenic cells.

Example 11 Expression of Potential Therapeutic Targets by the DifferentPopulations of Neoplastic Cells

In the deterministic solid tumor stem cell model (FIG. 1B), expressionof genes by the tumorigenic and non-tumorigenic populations of tumorcells may differ. Accordingly, the inability of current cancertreatments to significantly improve outcome is due to the tendency oftherapeutics to target non-tumorigenic cells but not the raretumorigenic solid tumor stem cells. If only the non-tumorigenicpopulations are killed by particular therapies, then tumors may shrink,but the remaining tumorigenic solid tumor stem cells can drive regrowthof the tumor.

We examined first passage T1 cells for expression of either the EGFreceptor (EGF-R) or HER2/neu. The EGF-R and HER2/neu are potentialtherapeutic targets that have been implicated in breast cancer cellproliferation. Tumorigenic T1 cells stained with lower levels ofanti-EGF-R antibody than non-tumorigenic cells, and EGF-R expressioncould not be detected at the single cell level in tumorigenic cells(FIG. 11).

To test whether cells that did not express detectable levels of theEGF-R were tumorigenic, 1,000-2,000 EGFR⁻B38.1⁺CD24⁻LINEAGE⁻ cells wereinjected into NOD/SCID mice. Tumors formed in four out of four cases,confirming that the EGF-R⁻ cells are tumorigenic. By contrast, we couldnot detect a substantial difference in HER²/neu expression betweentumorigenic and non-tumorigenic T1 cells (FIG. 11B and FIG. 11E).

As expected from the solid tumor stem cell model, 1,000-2,000HER²/neu⁺B38.1⁺CD24⁻LINEAGE⁻ cells gave rise to tumors in four out offour cases. This EXAMPLE shows that there can be differences in theexpression of therapeutic targets between the tumorigenic andnon-tumorigenic populations.

These experiments serve as a proof-of-principle of our stem cell modelof solid cancer and demonstrate the following:

(a) tumor cells are phenotypically and functionally heterogeneous;

(b) by separating cells by FACS one can enrich for tumorigenic cells;and

(c) by studying the tumorigenic fractions, one can isolate solid tumorstem cells to more carefully focus molecular and biological assays.

Example 12

Further Evidence for a Role for Notch in Breast Cell proliferation

To determine whether Notch was a solid tumor stem cell marker, we sortedNotch 4⁺ and Notch 4⁻ cells and analyzed their ability to form coloniesin vitro. Surprisingly, neither population grew in tissue culture.

There are two possible explanations for these results. First,interaction between the two populations of cells may be required forcell growth. Alternately, the antibody may be either an agonist orantagonist of Notch 4 and inhibition or activation of the receptor mayinhibit tumor cell growth. To distinguish between these possibilities,unseparated tumor cells were incubated with the anti-Notch 4 antibody,and then assayed for the ability to form colonies in vitro in tissueculture medium containing soluble Delta, a Notch ligand. Delta wasessential for colony formation from cells isolated from this tumor.While control cells grew in tissue culture and formed colonies (FIG.12A), cells incubated with the Notch 4 antibody did not grow (FIG. 12B).When the anti-Notch 4 antibody was pre-incubated with the peptide usedto generate the antibody (this peptide should theoretically blockbinding of the antibody to the cells), the ability of the tumor cells toform colonies was restored (FIG. 12C). An anti-Notch 2 antibody did notaffect colony formation.

To confirm that Notch regulates colony formation by tumor cells, cellswere incubated in medium with or without soluble Delta (FIG. 12A andFIG. 12D, respectively). The cultures with Delta formed many colonies(FIG. 12A), whereas only a few small colonies formed in medium lackingsoluble Delta or in cultures using cells exposed to the anti-Notch 4antibody (FIG. 12B, FIG. 12D, and FIG. 12E). Taken together, these datasuggest that the Notch 4 pathway regulates the proliferation/survival ofbreast cancer cells in cells and that the anti-Notch 4 antibody blocksactivation in vitro.

We also tested whether the anti-Notch 4 antibody inhibits breast cancertumor cell growth in vivo. To do this, 20,000, 10,000 or 5,000tumorigenic cells were incubated with the anti-Notch 4 antibody or theanti-Notch 4 antibody and the blocking peptide. The antibody delayed theappearance of the tumors by one week when either 20,000 or 10,000tumorigenic cells were injected, and by 13 days when 5,000 cells wereinjected. This suggests that the antibody may block tumor formation invivo.

We then tested four breast cancer cell lines for expression of Notch 4and growth inhibition by the anti-Notch 4 antibody. We found that theMCF-7 and the SKBR-3 breast cancer cell lines expressed Notch 4 and thatthe anti-Notch 4 antibody inhibited the growth of these cells. RT-PCRshowed that both expressed Notch 4. To determine whether the anti-Notch4 antibody inhibits growth, 5,000 MCF-7 cells were incubated with theanti-Notch 4 antibody, the anti-Notch 4 antibody and the peptide used togenerate the antibody, or an irrelevant control antibody. The anti-Notch4 antibody, inhibited colony formation of both cell lines by more thanten-fold.

This EXAMPLE shows that Notch 4 activation promotesproliferation/survival of solid tumor stem cells and that the anti-Notch4 antibody blocks receptor activation.

Example 13 Targeting Breast Cancer Stem Cells with a Gene TherapySuicide Vector

The breast cancer stem cell comprises a minority of the tumor cells in abreast tumor. It is an object of the invention that gene therapystrategies target these cells (FIG. 13).

We targeted virus vectors to breast cancer stem cells using markersexpressed by stem cells. To do this, we stained different cell lineswith the 838.1 antibody and analyzed them by flow cytometry. MCF7,SKBR3, T47D and BT474 breast cancer cell lines were highly positive. Wehad the anti-fiber and the B38.1 antibodies conjugated with the Prolinx(Prolinx, Inc., Bothell, Wash., USA) method (FIG. 14, see Douglas J T etal., Nature Biotechnology. 14(11):1574-8 (1996)). When we mixed themodified anti-knob and anti-B38.1 antibodies together, they becamecross-linked and generated the bi-specific conjugate (FIG. 15). Theanti-fiber antibody part of the conjugate binds to the adenovirus, whilethe anti-B38.1 moiety binds to the breast cancer stem cell. Incubationof the AdLacZ virus with the anti-fiber alone blocks the infectivity ofthe virus (FIG. 16). If we incubate the virus with the bi-specificconjugate, the infectivity is restored only in the cells that expresshigh levels of the B38.1 antigen (FIG. 18). The re-targeting isspecific, because it can be inhibited by free B38.1 antibody (FIG. 18).

The conclusion is that the new bi-specific conjugate modifies theinfectivity of Adenovirus, blocking its natural tropism and directingthe infection to cells that express the breast cancer stem cell surfacemarker.

Example 14 Downstream Targets in the Breast Cancer Stem Cells

The methods used in this EXAMPLE provide guidance for the development ofNotch-related and other anti-cancer therapies using the cancer stemcells of the invention. We use array technology to begin to understandthe molecular pathways that might be regulated by Notch-signalinginduced by specific Notch ligands. Sequence verified human cDNAs fromResearch Genetics, provided by the University of Michigan MicroarrayNetwork, are arrayed by the Cancer and Microarray Facility. Probes areprepared from self-renewing breast cancer stem cells or cells from thevarious populations of cells found in a tumor. Probes are hybridized tothe arrays and the hybridization patterns are read by the Cancer andMicroarray Facility. We then analyze the hybridization patterns toidentify genes that hybridize to probe from the breast cancer stem cellsstimulated with various Notch ligands and non-stimulated cells. Suchgenes may represent those that are involved in the regulation of breastcancer cell survival or self-renewal.

Preparation of microarrays. We have used the microarray technology toanalyze gene expression of hematopoietic stem cells. We now extend thiswork to cancer stem cells.

The University of Michigan Microarray Network currently has 32,500sequence verified human cDNAs from Research Genetics. A “cancer” chiphas been assembled in collaboration with the NCl. This chip contains acomprehensive constellation of 1,200 genes involved in proliferation andtumorigenesis. There is also an “apoptosis chip” developed by theUniversity of Michigan that contains all genes known to be involved inprogrammed cell death. Note that the HES genes, known to be downstreamtargets of Notch, are included in the arrays.

Preparation of probe from breast cancer stem cells. Messenger RNA isisolated either from freshly purified breast cancer stem cells or frombreast cancer stem cells incubated in the presence or absence of variousNotch ligands. The RNA is amplified if necessary, such as by PCR orlinear RNA amplification Wang et al., Nature Biotechnology. 18: 457-459(April 2000). Probe are prepared by reverse transcription from anoligo-dT primer, and labeled by incorporating CY3 or CY5 conjugatednucleotides. Gene expression profiles are examined using probe preparedfrom freshly isolated, uncultured breast cancer cells, as well as fromcultured breast cancer cells, such as cells that have been exposed tothe appropriate Notch ligands (including Fringe family members, eithersingly or in combination as determined by which ligands are expressed bythe different populations of tumor cells. To do these assays, we exposethe cells to a soluble form of Delta or Jagged family members in whichthe transmembrane region has been deleted, or one of the Fringes. TheFringes are secreted proteins. We make recombinant protein of each Notchligand of the Delta, Jagged and Fringe families from insect cells ormammalian cells transfected with a baculovirus or mammalian expressionvector, respectively.

Comparisons of gene expression patterns between control breast cancertumorigenic cells and tumorigenic cells exposed to various Notch ligandsare made. Probe from breast cancer stem cells from each tumor iscombined with probe labeled with a different fluor made from culturedbreast cancer stem cells exposed to various Notch ligands to comparetheir hybridization patterns. To do this, breast cancer stem cells areisolated by FACS. Cells are seeded at single cell density to precludeNotch interactions between cells. Cells are exposed to soluble forms ofDelta (see, EXAMPLE 6), Delta-like, Jagged 1, Jagged 2, or each of theFringes. Cells are exposed to each protein both alone and incombinations suggested by the Notch4 ligand expression pattern ofindividual cell populations. The microarrays hybridized with probe fromeach test condition are compared and analyzed to gain insights intomolecular pathways affected by Notch ligand interactions. For example,if a particular population of cells expresses Delta and Manic Fringe,then one group of breast cancer stem cells is exposed to Delta alone, asecond to Delta and Manic Fringe and a third to Manic Fringe alone. cDNAis made from each population with Cy5 or Cy3 labeling, and used to probea microarray chip. In addition, cDNA from each population is used withcDNA made from cells cultured in control medium and freshly isolatedbreast cancer cells to probe a microarray chip. Each group is compared 5times to assure that any differences in expression profiles of thearrayed genes by each test groups are real.

Preparation of probe from cells treated with the anti-Notch 4 antibody.An antibody against Notch 4 inhibits growth in vitro and tumorigenesisin vivo. This effect could be explained if the antibody acts as either aNotch-4 agonist or antagonist. Since soluble Delta promotes cancer cellgrowth in vitro, the antibody most likely is a Notch 4 antagonist. Toconfirm the mechanism by which the anti-Notch 4 antibody inhibits tumorgrowth, probe is made from cells incubated in the presence or absence ofthe anti-Notch 4 or control irrelevant antibody and the variouscombinations of the Notch ligands and used for microarray expressionanalysis as described above. Another control group includes cellsincubated with the antibodies and no Notch ligand. Each comparison isperformed in at least six independent tests employing independentlyprepared batches of probe. By comparing the gene expression patterns ofeach group, we should be able to determine how the anti-Notch 4 antibodyaffects Notch signaling.

Making the cDNA probe. 1-2 μg of mRNA is commonly used to synthesizeprobe for screening gene expression profiles on microarrays (Wang etal., Nature Biotechnology. 18: 457-459 (April 2000)). Since we screen aset of three slides (containing a total of 32,500 cDNAs) in each test, 6μg of mRNA is required per assay (reverse transcription of 6 μg of mRNAshould yield around 3 μg of cDNA probe, or 1 μg of probe per slide).Cancer cells tend to have a high RNA content. In past assays, 10⁷ cancercells yielded around 100 μg of total RNA, which in turn yielded around 3μg of poly A⁺RNA. Thus in order to generate 6 μg of mRNA, around 2×10⁷cells would be required. As described in the preliminary data, thatnumber of flow-cytometrically purified breast cancer stem cells can beisolated from approximately five-ten 1 cm tumors.

The breast cancer stem cells represent approximately 5% of the totalnumber of cells within a tumor. It is not practical to isolate more than10⁶ freshly dissociated (uncultured) breast cancer stem cells byflow-cytometry in one day. This would yield less than 0.5 μg of mRNAfrom one day of sorting. While breast cancer stem can be combined frommultiple days of sorting to pool enough mRNA to prepare probe fromfreshly isolated cells, it may not be practical to perform all assays inthis manner. Some assays require brief periods of tissue culture.Plating efficiency of the sorted cells is approximately 10%. Thus it maybe necessary to enzymatically amplify the template prior to synthesizingprobe. This can be done either by PCR or by linear amplification of RNAusing T7 RNA polymerase. Our protocol employs 15-18 rounds of PCR toamplify cDNA from small numbers of stem cells. This protocol was used toconstruct a high quality hematopoietic stem cell (HSC) cDNA library andto make probe from hematopoietic stem cells. To produce probe fromfreshly isolated breast cancer stem cells, we test the same approach.Alternately, a number of groups have reported success in using linearRNA amplification to produce probe for microarray hybridization. Thus wecompare the two methods by preparing probe both ways and examining thehybridization patterns that result. cDNA is primed using an oligo-dTprimer that contains a T7 RNA polymerase binding site and synthesized bySuperscript reverse transcriptase (Gibco) and the Clontech 5's switcholigomer that allows the tagging of the 5′ end of the cDNA. Secondstrand cDNA is synthesized using E. coli DNA polymerase. Then amplifiedRNA (aRNA) is produced using T7 RNA polymerase or PCR. Which of the twothat amplification methods are used is determined by comparing probemade with standard cDNA synthesis. After preparing aRNA, cDNA isre-synthesized using random hexamers. This cDNA can then be used forprobe, or if necessary, additional rounds of amplification can beperformed. Both approaches are used to prepare probe from 40,000 MCF-7cells (a human breast cancer stem cell line). This probe is hybridizedto human cDNA microarrays along with probe from unamplified MCF-7 cells.The amplification approach that most closely reproduces thehybridization pattern of the unamplified probe is selected. Thenamplification conditions are modified until the amplified probereproduces the hybridization pattern of the unamplified probe as closelyas possible.

Analysis of the hybridization pattern. Hybridization patterns areanalyzed in the Cancer and Microarray Core facility using their laserscanning system. The use of an integrated system for arraying,hybridizing, scanning, and analyzing hybridization patterns in which allcomponents are provided by Genomics Solutions permits a seamless andefficient analysis of hybridization patterns.

A transcript is differentially expressed if there is at least a 3-folddifference in normalized hybridization levels between probes.Hybridization signals from Cy3 and Cy5 labeled probes within a singletest are normalized to each other to correct for potential differencesin the effective concentration of each probe and replicates of each testare done using the opposite fluor for each group to correct fordifferences in the amounts or labeling efficiencies of probes.

Verification of differential expression. cDNAs that consistentlyhybridize to probe from groups of cells but not to probe from thecontrol groups of cells are further characterized. The sequences ofthese cDNAs are obtained from the Microarray Network. Two approaches areused to confirm the differential expression of candidate genes betweencell populations. The first is to prepare in situ hybridization probesagainst candidate genes, and then perform in situ hybridizations onbreast cancer stem cells cultured in medium with or without variousNotch ligands as described above. In situ hybridizations are thenperformed on cultured cells. The advantage of this approach would bethat expression could be compared at the level of individual cells.

An alternate approach is to design nested PCR primers against candidategenes, and to perform RT-PCR on multiple 1-10-cell aliquots of freshlypurified breast cancer stem cells (isolated as described above). Byperforming RT-PCR on small numbers of cells it is possible to observe adifference in the ability to amplify particular transcripts, even if the“non-expressing” population contains rare expressing cells. We used thisapproach to demonstrate the differential expression of RGS18 betweendifferent subpopulations of multipotent hematopoietic progenitors.

Differential expression can be confirmed by Northern analysis. PolyA⁺RNA from 1-2×10⁷ breast cancer stem cells cultured with or without theNotch ligands, are hybridized to probes of the differentially expressedcDNAs. Hybridization signals are quantitatively compared between thesesamples.

We then confirm that genes are differentially expressed at the proteinlevel. In cases where immunocytochemical staining is uninformative wealso perform western blots on protein from the different cells.

Certain molecular analyses are difficult using the primary breast cancercells that only proliferate for prolonged periods of time in thexenograft model. These analyses can be done in cell lines. One can useany of a large number of breast cancer cell lines, including earlypassages of lines that we have studied in the past. Clarke et al., Proc.Natl. Acad. Sci. USA. 92: 11024-28 (November 1995); Hernandez-Alcocebaet al., Human Gene Therapy. 11(11): (September 2000). These cell linesare plated at single-cell density with and without various Notchligands, as well as the anti-Notch 4 or control antibodies as describedin the assays with the primary breast cancer cells. If clonogenicity isaffected by Notch signaling, then probe for the microarray analysis ismade using cDNA made form the cell line incubated in medium with orwithout the various Notch ligands or anti Notch 4 or control antibodies.Since a virtually unlimited number of cells can be analyzed, we can makeprobe that has not been amplified.

Finally, cell lines are useful for confirming whether the anti-Notch 4antibody is an agonist or antagonist. If a cell line is identified thatclonogenicity is enhanced by soluble Delta and inhibited by theanti-Notch 4 antibody, then it is used in these assays. The cells arestably transfected with a luciferase minigene under the control of theNotch-inducible HET-1 promoter. Jarriault et al., Molecular & CellularBiology. 18(12): 7423-31 (December 1998). The cells are plated at singlecell density to prevent cell-cell Notch-Notch ligand interactions. Theyare treated with the various combinations of Notch ligands and eitherthe anti-Notch 4 antibody or a control antibody. The cells are harvestedand a luciferase assay is done to determine how each condition affectsNotch signal transduction as reflected by transactivation of the HETIpromoter.

A comprehensive functional analysis of candidate genes that emerge fromthe microarray analysis can be performed. Full-length cDNAs are isolatedand cloned into a retroviral expression vector. Breast cancer cell linesand breast cancer stem cells isolated from the five xenograft tumors areinfected in vitro and the effect of the retroviral transgene onself-renewal and tumorigenicity is assayed relative to clones infectedwith a control vector. The transgene is expressed as a bicistranicmessage that contains IRES-GFP. This allows identification of transducedcells via FACS or fluorescent microscopy. The effect of the transgene onNotch signaling is examined in vitro and in vivo. To do this, transducedcells are tested for response to the various combinations of Notchligands found to affect colony formation in tissue culture andtumorigenicity in mice.

The expression patterns of candidate genes are examined in detail invivo to determine how widely the genes are expressed beyond thexenograft. In addition to performing more extensive in situhybridizations of tissue sections from slices of primary breast cancer,we make antibodies against selected gene products being studied. TheHybridoma Core facility at the University of Michigan has extensiveexperience preparing monoclonal antibodies using both peptides, andexpressed recombinant proteins.

Ultimately, the functions of unknown genes are tested in vivo, usinggene targeting to make knockout mice. The University of MichiganTransgenic Core has established murine ES cell technology, they provideES cells that ‘go germline’ at a high rate and assist with thegeneration of homologous recombinant ES clones.

The ability of microarray analysis to simultaneously compare theexpression of many genes provides unparalleled power to screen forchanges in gene expression patterns. Combined with the ability to purifystem cells and to regulate their self-renewal and differentiation invitro, microarray analyses can be applied with great precision to screenfor specific types of regulatory genes.

Example 15 How Notch Signaling Affects Breast Cancer Tumorigenesis

To verify the importance of Notch in normal mammary growth anddevelopment, we test the effects of Notch ligands or agonist peptides onthe growth and tumor formation of mammary tumor stem cells.

Effect of Notch activation on tumor formation in vivo. The effects ofthe various combinations of Notch ligands on tumor formation arecorrelated with the microarray data obtained in EXAMPLE 14. These assaysare done using xenograft tumor cells before and after enrichment ofpopulations of malignant breast cells, allowing us to differentiateNotch effects on both stem and progenitor cells respectively. Theeffects of Notch ligands on growth and differentiation of eachpopulation of cancer cells are determined utilizing the in vivo assay.

To determine the effect of Notch stimulation on tumor growth in vivo,one thousand, ten thousand, and one hundred thousand breast cancer tumorcells are incubated with soluble Notch ligands, alone or in combination,or control media and then injected (6 replicates of each) into NOD/SCIDmice. The soluble forms of Delta, Delta-like, Jagged 1, and Jagged 2 areused as well as members of the Fringe family. The cells are incubatedwith each ligand individually, as well as combinations of differentligands, for two hours. The cells are suspended in Matrigel™ with theNotch ligands and then injected into the mice. The effects of eachligand of Notch stimulation on both the number of cells needed to form atumor as well as the time needed to form a tumor are determined. Thisallows us to determine whether Notch affects tumor growth in vivo.

In previous EXAMPLES, the anti-Notch 4 antibody retarded tumor formationafter a single, brief incubation with cells. Next, one thousand, tenthousand, and one hundred thousand tumorigenic cells from each of thefive xenograft tumors are incubated with either a control antibody orthe anti-Notch 4 antibody alone, or with various combinations of Delta,Jagged and Fringe family proteins. Each group is then mixed in Matrigel™containing the same antibody or Notch ligands and injected into mice.The effects of the anti-Notch 4 antibody on the number of cells and timeneeded to form a tumor are measured. The results are correlated with themicroarray expression data, obtained in EXAMPLE 14.

Stimulation of Notch should drive the proliferation of the normal breaststem cells. Like in normal stem cells, members of the Fringe familyshould modulate Delta or Jagged signaling. Notch should also play a rolein the self-renewal of the breast cancer stem cell.

Example 16 Human Subjects

The following human sources of material are obtained for use in themethods of diagnosis of the invention: (1) primary tumors from patientswith breast cancer and (2) pleural fluid from patients with metastaticbreast cancer. A portion of the tumor or the pleural fluid is obtainedduring routine treatment when the patient's physician deems removal ofthe tumor or pleural fluid to be clinically indicated.

For example, at the University of Michigan Hospital, there is an IRBapproved protocol to obtain the specimens. Patients are asked to sign aninformed consent where indicated. There are no additional risks topatients.

Example 17 Vertebrate Animals

The University of Michigan complies with the Animal Welfare Act asamended (7 U.S.C. §2131 et seq.), and other Federal statutes andregulations relating to animals. We use the laboratory mouse strainNOD/SCID mice and other strains of immunocompromised mice, such asBeige/SCID mice.

There should not be any discomfort from cancer cell growth, because miceundergo euthanasia prior to development of illness. If the mice do showevidence of discomfort from tumors (posture, appetite, or behaviorchanges, weight loss), they can be immediately sacrificed. Should themice show evidence of more than mild discomfort (posture, appetite, orbehavior changes) they are given analgesics such as oral morphine orcodeine. Mice have bone marrow cells inoculated into their retro-orbitalvein while anaesthetized with either ether or a similar generalanesthetic agent.

Mice are killed by the use of CO₂, which is consistent with therecommendations of the Panel on Euthanasia of the American VeterinaryMedical Association. This method was chosen because it is safe andhumane.

Example 18 Cell Cycle Analysis of Breast Cancer Stem Cells andNon-Tumorigenic Breast Cancer Cells

We tested cells from a mouse xenograft tumor. The cell cycle status ofthe breast cancer stem cell (FIG. 19A) and non-tumorigenic populationsof cells (FIG. 19B) in the tumor was determined by flow cytometry. Therewas a similar distribution of cells in the G1, S, and G2/M phases of thecell cycle in both populations. Thus, both the tumorigenic andnon-tumorigenic populations exhibited similar cell-cycle distributions.

This EXAMPLE rules out the possibility that all cells that expressed thesolid tumor stem cell markers were at a particular stage of the cellcycle in the tumor that was analyzed. By selecting cells at a particularstage of the cell-cycle by flow-cytometric sorting of Hoechst stainedtumor stem cells it may be possible to further enrich tumorigenicactivity, just as it has been possible to enrich hematopoietic stem cellactivity by selecting cells with low levels of Hoechst staining

Example 19 Rhodamine 123 Staining of Breast Cancer Stem Cells andNon-Tumorigenic Breast Cancer Cells

The purpose of this EXAMPLE is to examine the activity of the multi-drugresistance pump in breast cancer stem cells and the non-tumorigeniccancer cells. We have stained the ESA⁺CD44⁺CD24^(−/lo)LINEAGE⁻ cells(breast cancer stem cells) and the non-tumorigenic cells obtained fromone of the xenograft tumors with Rhodamine 123.

One of the major factors that determines the intensity of Rhodamine 123staining in a cell is the MDR pump activity that eliminates this dyefrom the cells. Yumoto R. et al., Drug Metabolism & Disposition 29(2):145-51 (2001); Daoud R. et al., Biochemistry 39(50): 15344-52 (2000). Wefound that some of the solid tumor stem cells from this tumor stainedless intensely with Rhodamine 123 than did the non-tumorigenic cancercells (FIG. 20).

This EXAMPLE shows tumor cell heterogeneity and indicates that MDR pumpactivity may be higher in a solid tumor cells.

Example 20 A B38.1⁺CD44⁺CD24^(31/lo)Lineage⁻ Population of Cells Existsin Ovarian Cancer Tumors

We analyzed cancer cells obtained from both a tumor and ascites fluidobtained from a cyto-reduction surgery for a patient with ovariancancer. Notably, B38.1 is known to be expressed by ovarian cancer cells.Surprisingly, the flow cytometry analysis revealed multiple cellpopulations, and there was a distinct B38.1⁺CD44⁺CD24^(−/lo)LINEAGE⁻population of cells (FIG. 21).

This distinct cell population phenotypically resembles the breast cancerstem cell and may represent an ovarian cancer stem cell.

Example 21 A B38.1⁺CD44⁺CD24^(−/lo)Lineage⁻ Population of Cells Existsin Sarcoma Tumors/Comparison to Breast Cancer Stem Cell Data

The B38.1 antigen had previously been described to be expressed only inbreast cancer and ovarian cancer. We established two sarcomas by placingsarcoma cells in the flanks of NOD/SCID mice that had been treated withVP16. One of the tumors was excised and examined by flow cytometry.Surprisingly, the sarcoma cells expressed the B38.1 antigen.Furthermore, there were three distinct cell populations with respect toCD44: high, low and negative (FIG. 22).

Thus sarcoma cells include a CD44+ population that phenotypicallyresembles breast cancer stem cells and that may represent sarcoma stemcells.

TABLE 7 Tumorigenicity of Different Populations of Tumor T1 and T2 Cells# tumors/# of injections Cells/injection 8 × 10⁵ 5 × 10⁵ 2 × 10⁵ T1cells CD44⁻ 0/2 0/2 — CD44⁺ 2/2 2/2 — B38.1⁻ 0/2 0/2 — B38.1⁺ 2/2 2/2 —CD24⁺ — — 1/6 CD24⁻ — — 6/6 T2 cells CD44⁻ 0/2 0/2 — CD44⁺ 2/2 2/2 —B38.1⁻ 0/2 0/2 — B38.1⁺ 2/2 2/2 — CD24⁺ — — 1/6 CD24⁻ — — 6/6 Cells wereisolated by flow cytometry as described in FIG. 2 based upon expressionof the indicated marker and assayed for the ability to form tumors afterinjection of 2-8 × 10⁵ cells into the mammary fat pad of NOD/SCID mice.The number of tumors that formed/the number of injections that wereperformed is indicated for each population of cells.

Example 22 Preparation of Polynucleotide Probes

Single round RT labeling from total RNA source. The polynucleotidesisolated from isolated solid tumor stem cells or enriched populations ofsolid tumor stem cells can be RNA extracted from the cells orcomplementary DNA (cDNA) made from the extracted RNA.

For some embodiments of the methods of the invention (such as thoseinvolving hybridization), labeled cDNA is useful. In one embodiment, weuse the methods of single round RT labeling from total RNA source.

The quality of the RNA coming into a labeling reaction will have amarked effect on the quality of the hybridization. RNA preparations thatlook good by the standard molecular biology criteria can give poorresults. Typical problems include disperse, fine-grain noise over theentire hybridized surface and non-specific binding of fluor to the zonesof DNA immobilization on the slide. These problems seem likely to havesome roots in contaminating carbohydrate, and as would be expected withcarbohydrate, the problems are exacerbated by ethanol precipitationsbefore and after labeling. Very impure preparations frequently producevisible aggregates if precipitated after labeling and ethanolprecipitation, which are essentially resistant to solubilization. It iswell known that nucleic acids form strong aggregates with carbohydratewhen either dried together or when co-precipitated. This interaction isthe basis for nucleic acid “immobilization onto chromatography supportssuch as cellulose. To minimize this sort of problem, we recommend”preparative procedures which use few or preferably no ethanolprecipitations during RNA preparation and labeling. We use at least thevolumes of extractant and washing solutions suggested for” the number ofcells being processed. Appropriate or slightly excessive extraction/washvolumes tend to minimize noise in a hybridization assay.

Methods that give satisfactory results are Triazol extraction (BRL) andRneasy (Quiagen).

Total RNA is prepared from tissue (from solid tumor or xenograft tumor),tissue culture or enriched populations of solid tumor stem cellsobtained by flow cytometry. We resuspend the prepared RNA in a volumethat produces an RNA concentration of >6 mg/ml in DEPC water. If the RNAis recovered from a matrix as the final preparative step, and is stilltoo dilute, we concentrate as needed for labeling, using a MicroCon 30(Amicon) determine the concentration of the RNA. We test by reading asmall sample (A260) in 50 mM NaOH.

For the nucleotide mix, we use 10× low T dNTPs (using 100 mM dNTPs fromPharmacia (St. Louis, Mo., USA) [27-2035-02])

TABLE 8 NUCLEOTIDE MIX Nucleotide μl mM final (1/10) concentration dGTP25 0.5 dATP 25 0.5 dCTP 25 0.5 dTTP 10 0.2 water 415 total volume 500

Fluorescent Nucleotides are from Amersham Life Sciences or Perkin ElmerApplied Biosystems Division. We use FluoroLink Cy3-dUTP (#401-896) orFluoroLink Cy5-dUTP. The Cy 3 and Cy 5 nucleotides come at concentrationof 1 mM.

The R110 nucleotide comes at concentration of 0.1 mM, and must be driedand resuspended at 0.1× the initial volume to bring it to 1 mM.Currently, the factors of labeling efficiency, fluorescent yield,spectral separation, and tendency toward non-specific binding make theCy3/Cy5 pair the most useful for the detection system.

For the labeling reaction, the primer can be Pharmacia oligo (dT) 12-18(27-7858-01).

Unlabeled nucleotide mixes are prepared from Pharmacia 100 mM stocks.The reverse transcriptase is BRL SuperScript II (18064-014). The 5×buffer is the buffer supplied with the polymerase.

For use in microarrays, normalization in a two fluor labeling isachieved by reference to housekeeping genes distributed through themicroarray.

A cocktail of synthetic cDNAs produced using phage RNA polymerases oncloned E. coli genes in the pSP64 poly(A) vector as a mass standard andRNA quality standard. If possible, we also prepare a total RNA solutioncontaining 100 μg of RNA in 17 μl of DEPC water. Otherwise, we ethanolprecipitate 100 mg of total RNA to concentrate sample for labelingreaction. We take care to remove all residual 70% ethanol wash either byair drying or vacuum, as residual ethanol may impede the efficiency oflabeling.

We resuspend the pellet in DEPC water to give a final volume of 17 ml.Any residual precipitate is removed by centrifugation. The RNA is addedto the reaction last.

Fluor nucleotide (NTP) RT labeling: We add the following:

TABLE 9 REVERSE TRANSCRIPTASE LABELING Component μl 5× first strandbuffer 8 Oligo (dT) 12-18 (500 μg/ml) 2 10X low dT NTP mix 4 Fluor dUTP(1 mM) 4 0.1M DTT 4 RNAsin 1 syn mRNA std (0.06 μg) 0.5 100 mg total RNA17.0 total 40

We vortex the sample after adding RNAsin. We minimize bubbles andfoaming during the vortex or quick spin.

Next, we hold at 65° C. for 5 minutes, bring to 42° C. (program R1).Then, add 2 μl of SSII enzyme. Make sure enzyme is well mixed into thereaction. Next, incubate 42° C. for 25 minutes. Add 2 μl of SSII enzyme.Make sure enzyme is well mixed into the reaction. Then, incubate 42° C.for 35 minutes. Add 5 μl of 500 mM EDTA. Be sure to stop the reactionwith EDTA before adding NaOH.

Add 10 ml of 1M NaOH. Incubate at 65° C. for 60 minutes to hydrolyzeresidual RNA. Cool to room temperature and add 25 μl of 1 M Tris-HCl (pH7.5)

For probe cleanup and analysis, we transfer to a Microcon 30,concentrate to about 20 μl (approx 3.5 min at 14,000 rpm in Eppendorf5415C). We wash by adding 200 μl of TE (pH 7.5) and concentrating toabout 20 μl (4-4.5 min @ 14,000 rpm). We recover the product byinverting the concentrator over a clean collection tube and spinning for3 min @ 3000 rpm.

In some cases, the Cy5 probe may produce a gelatinous blue precipitatewhich is recovered in the concentrated volume. The presence of thismaterial signals the presence of contaminants. The more extreme thecontamination, the greater the fraction of probe which will be capturedin this gel. Even if heat solubilized, this material tends to produceuniform, non-specific binding to the DNA targets.

When concentrating by centrifugal filtration, the times required toachieve the desired final volume are variable. Overly long spins canremove nearly all the water from the solution being filtered. Then, whenfluor tagged nucleic acids are concentrated onto the filter in thisfashion, they are very hard to remove. Thus, we approach the desiredvolume by conservative approximations of the required spin times.

We take a 2 μl aliquot of Cy5 probe for analysis, leaving 17-18 μl forhybridization, then run this probe on a 2% agarose gel in TAE (gel sizeis 6 cm wide×8.5 cm long, 2 mm wide teeth). For maximal sensitivity whenrunning samples on a gel for fluor analysis, we use loading buffer withminimal dye and do add ethidium bromide to the gel or running buffer.

We scan the gel on a Molecular Dynamics Storm fluorescence scanner(Settings—red fluorescence, 200 micron resolution, 1000 volts on PMT). Asuccessful labeling produces a dense smear of probe from 400 bp to >1000bp, with little pile-up of low molecular weight transcripts. Weaklabeling and significant levels of low molecular weight materialindicate a poor labeling.

Hybridization. The blocking species is poly(dA) from Pharmacia(27-7988-01) (resuspended at 8 mg/ml); yeast tRNA from Sigma (R8759)(resuspended at 4 mg/ml); and CoT1 DNA from Life Technologies Inc.(concentrated 10 fold to 10 mg/ml).

The volume required for the hybridization is dependent on the size ofarray used. For hybridizations that require small volumes (20-25 μl),the probe volumes after microcon concentration can be too large. If so,then we add the blockers to the Cy3 probe and precipitate the Cy3 probe.

We add 8 mg poly (dA); 4 mg tRNA; and 10 mg CoT1 DNA per 10 mlhybridization.

TABLE 10 HYBRIDIZATION PROBE MIX Cy3 labeled probe ~20 μl   poly dA (8mg/ml) 1 μl yeast tRNA (4 mg/ml) 1 μl CoT1 DNA (10 mg/ml) 1 μl

We add 2 μl of 3 M sodium acetate (pH 5.5), then add 60 μl of ethanol,centrifuge, dry lightly, and resuspend in the }˜17 μl of Cy5 probe. Ifthe array requires approximately 40 μl of probe, then the Cy3 and Cy5concentrates are pooled and the blockers are added directly, so noprecipitation is required.

Then, we add 3 μl of 20×SSC per 20 μl of hybridization mix volume. Atthis point, we optionally add 1 μl of 50×Denhardt's blocking solutionper 20 μl of hybridization mix. With very clean probe, the Denhardt'sdoes not make any visible difference.

Then, we heat at 98° C. for 2 minutes, cool to 45° C. and add 0.2 μl of10% SDS per 20 μl of hybridization mix volume, then apply to the array,and hybridize (16-24) hours at 65° C. in a sealed, humidified chamber.

Washing. Residual unbound probe is removed from the slides by washing2-5 minutes each at room temperature. The first wash is 0.5×SSC, 0.01%SDS. The second wash is 0.06×SSC. Air drying of the slides after thisstep can leave a fluorescent haze on the slide surface, so buffer isremoved from the slides by a brief spin at low G. We place the slides ina slide holder and spin in a centrifuge equipped with a swinging carrier(horizontal) which can hold the slide holder. Most centrifuges that areadaptable to centrifuging microtiter plates can be used for thispurpose.

Example 23 Cancer Stem Cells Display Elevated Levels of ALDH Activity,Accounting for CPA-Treatment Cell Resistance Via Increased Cancer StemCell Frequency

Cancer stem cells were subsequently isolated from colon cancers (U.S.application Ser. No. 11/591,019, herein incorporated by reference). Todetermine whether colon tumor cells contain ALDH activity, primaryxenograft tumors depleted of murine cells (using antibodies against CD45and H2K^(d)) were screened using the Aldefluor™ reagent. The majority ofCD44⁺ cells, approximately 85%, contained ALDH activity levelsdetectably higher than the bulk of colon tumor cells (FIG. 23).Consistent with this observation, microarray analyses indicated thattumorigenic UM-C4, UM-C6, and OMP-C9 TG cells express several ALDHfamily members (e.g. ALDH1A1, ALDH1B1, ALDH2, ALDH3A2, ALDH5A1, ALDH6A1,ALDH7A1, ALDH9A1 & ALDH18A1) at higher levels than NTG populations(FIGS. 24A & B). Following isolation of ALDH and CD44 subpopulationsfrom UM-C4 tumors by dissociation and FACS, only the ESA⁺44⁺ALDH⁺subpopulation contained tumorigenic ability upon transplantation (FIG.23B). In contrast to ESA+CD44+ALDH+ cells from UM-C4 colon tumors,tumorgenic cells from other xenogenic tumor lines (i.e. UM-C6, OMP-05,and OMP-C8) did not co-purify with ALDH activity (FIG. 23C; Table 11), aresult that may reflect heterogeneity among patients, disease stage, thetumorigenic cell of origin, and/or the repertoire of ALDH geneexpression.

TABLE 11 Tumors/Injections 2000 1000 500 250 200 100 40 UM-C4 ESA⁺CD44⁺5/5  11/12 17/20 — 29/33 10/10 11/32 ESA⁺CD44⁺ 0/20  2/24 0/4 —  0/20 —— ESA⁺CD44⁺ALDH⁺ — — 5/5 5/7 — — — ESA⁺CD44⁺ALDH⁻ — — 0/5 0/6 — — —ESA⁺CD44⁻ALDH⁺ — — 0/5 0/6 — — — UM-C6 ESA⁺CD44⁺ 4/4  5/5 10/10 —  7/11— — ESA⁺CD44⁻ 0/10  0/10 — — 0/4 — — ESA⁺CD44⁺ALDH⁺ — —  3/13 2/9 — — —ESA⁺CD44⁺ALDH⁻ — — 2/2 2/8 — — — ESA⁺CD44⁻ALDH⁺ — —  0/10 0/7 — — —OMP-C5 ESA⁺CD44⁺ 4/5  —  7/10 — — — — ESA⁺CD44⁻ 0/10 — — — — — —ESA⁺ALDH⁺ — — 3/9 — — — — ESA⁺ALDH⁻ — —  1/11 — — — — OMP-C8 ESA⁺CD44⁺5/5  4/4  5/10 — — —  2/10 ESA⁺CD44⁻ 1/15 — — — — — — ESA⁺CD44⁺ALDH⁺ — — 5/11 — — — — ESA⁺CD44⁺ALDH⁻ — —  6/13 — — — — ESA⁺CD44⁻ALDH⁺ — — 0/2 —— — —

Interestingly, the ALDH3A1 gene product may also mediate resistance toCPA, and its expression was uniquely elevated in microarray studies ofUM-C4 tumorigenic versus non-tumorigenic cells, but not differentiallyexpressed in UM-C6 or UM-C9 cells (FIGS. 24A & B). To validatemicroarray data, RNA was independently isolated from FACS-purified coloncancer stem cells and non-tumorigenic cells, and relative geneexpression was assessed using Taqman assay quantitative RT-PCR. ALDH1A1was validated as being dramatically increased in TG versus NTG cells(FIG. 24C).

Cyclophosphamide (CPA) promotes DNA cross-linking and is a commonly usedchemotherapeutic agent, against which patients frequently developresistance. The elevated levels of ALDH and expression of a number ofALDH family member genes in tumorigenic colon tumor cells couldcontribute to such resistance in the very cells responsible for tumorrecurrence post-treatment. To test this, the resistance of tumorgeniccells from UM-C4 tumors with high ALDH activity to CPA therapy comparedto the majority of non-tumorigenic tumor cells (CD44^(neg) and/orALDH^(neg)) was determined. Following tumor initiation with 500ESA⁺CD44⁺ cells, tumors were allowed to reach ˜400 mm³, at which pointthe mice were randomized to receive either 25 mg/kg CPA or vehicle twiceweekly. UM-C4 tumor growth in CPA-treated mice was retarded versus micetreated with vehicle (FIG. 25A). Furthermore, ESA⁺CD44⁺ cells were moreconcentrated in tumors from CPA-treated versus vehicle-treated mice(FIG. 25B) and the frequency of cells with high ALDH activity wasincreased >70% (FIG. 25C), suggesting that tumorgenic cells were trulymore refractory to therapy than non-tumorgenic cells.

To determine whether the increase in ESA⁺CD44⁺ALDH⁺ cells in CPA treatedtumor compared to control vehicle-treated controls correlated with anincrease in tumorgenic cell frequency, limiting doses of residual tumorcells were transplanted into naïve NOD/SCID hosts. Specifically, todetermine the frequency of cancer stem cells in CPA-treated versusvehicle-treated animals, four sets of eight to more mice receivedtransplantation of 3-fold serial dilutions of bulk tumors cells,starting with 1,500 cells (dilutions: 1500, 500, 167 & 56 cells). Micewere monitored for tumor growth over a period of four months and scoredas either positive or negative for tumor growth such that Poissonstatistics could be done to assess tumorgenic cell frequency among theinput population. All eight mice injected with 1,500 cells from eitherCPA-treated or vehicle-treated tumors developed tumors (Table 11).

Input of less cells clearly revealed an increased frequency oftumorgenic cells in tumors from CPA-treated mice, as 9/9, 5/8 and 4/8mice developed tumors with an input of 500, 167 and 56 cell,respectively; whereas 6/8, 3/8 and 2/8 mice grew tumors from seriallytransplanted vehicle-treated tumors. These frequencies translated into a1:120 versus 1:315 frequency of tumorgenic cells in the bulk populationof CPA-treated versus control vehicle-treated tumors, demonstratinga >2.6-fold increase in tumorigenic cells in CPA-treated tumors (FIG.25D; p=0.024). Finally, though the inherent ability of ESA⁺CD44⁺ cellsto generate tumors appeared equal among CPA-treated and vehicle-treatedtumors, the data show a trend towards more aggressive tumor growth withcells sorted from CPA-treated tumors (FIG. 25E).

Example 24 Anti-Integrin Beta 1 Treatment Reduces the Number of CancerStem Cells

A limiting dilution assay was used to assess the effect of anti-integrinbeta 1 antibody treatment on solid tumor cancer stem cells. Treatmentwith AIIB2 antibodies (Developmental Studies Hybridoma Bank (DHSB),University of Iowa) reduced the percentage of colon cancer stem cellsrepassaged into immunocompromised mice (FIG. 26).

C17 colon tumors from mice treated with either control or AIIB2antibodies at 10 mg/kg twice per week were isolated after 38 days oftreatment. Isolated tumors (n=3 per experimental group) were dissociatedand FACS analyzed to determine the ratio of mouse to human cells. Humancells in increasingly lower numbers were then re-injected intoimmuno-compromised mice. Specifically, mice were injected with 1000,333, 111, or 37 isolated human tumor cells in the right flank region(n=10 per group). Tumor volume was assessed twice per week. At day 28(D28) the percentage of mice with detectable tumors was decreased in allgroups injected with AIIB2 treated tumor cells compared to controls(FIG. 26A): Ten out of 10 mice injected with 1000 control antibodytreated tumor cells had detectable tumors compared to 8 out of 10 miceinjected with 1000 AIIB2 antibody treated tumor cells. Ten out of 10mice injected with 333 control antibody treated tumor cells haddetectable tumors compared to only 1 out of 10 mice injected with 333AIIB2 antibody treated tumor cells. Eight out of 10 mice injected with111 control antibody treated tumor cells and 7 out of 10 mice injectedwith 37 control antibody treated tumor cells had detectable tumorscompared to none of the mice injected with 111 or 37 AIIB2 antibodytreated tumor cells.

Similarly on day 34 (D34), animals injected with 333, 111, and 37 AIIB2treated tumor cells showed reduced numbers of tumors compared tocontrols (FIG. 26B). Specifically, 10 out of 10 mice injected with 333control antibody treated tumor cells had detectable tumors compared to 7out of 10 mice injected with 333 AIIB2 antibody treated tumor cells. Tenout of 10 mice injected with 111 control antibody treated tumor cellshad detectable tumors compared to only 3 out of 10 mice injected with111 AIIB2 antibody treated tumor cells. And finally, 10 out of 10 miceinjected with 37 control antibody treated tumor cells had detectabletumors compared to only 1 out of 10 mice injected with 37 AIIB2 antibodytreated tumor cells.

Even after 42 days (D42), animals injected with 111 and 37 AIIB2 treatedtumor cells showed reduced numbers of tumors compared to controls (FIG.26C). Specifically, 10 out of 10 mice injected with 111 control antibodytreated tumor cells had detectable tumors compared to only 4 out of 10mice injected with 111 AIIB2 antibody treated tumor cells. And 10 out of10 mice injected with 37 control antibody treated tumor cells haddetectable tumors compared to only 2 out of 10 mice injected with 37AIIB2 antibody treated tumor cells.

The stem cell frequency was calculated using L-Calc™ software (StemcellTechnologies). Briefly, based on Poisson statistics, exactly one stemcell exists among the known number of injected cells if 37% of theanimals fail to develop tumors. Treatment of tumors with anti-integrinbeta 1 antibodies reduced the stem cell frequency from 1:49 to 1:1,179.The 95% confidence intervals range from 1:87 to 1:27 for control treatedcells compared to 1:2,241 to 1:621 for AIIB2 treated cells (FIG. 26D).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described compositions, methods, systems, and kits of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in art are intended to be within the scope of thefollowing claims.

1. A method for determining the capability of a test compound to affectthe number of solid tumor stem cells from a solid tumor of epithelialorigin, the method comprising: a) exposing a first set of the tumorcells from a solid tumor of epithelial origin, but not a second set ofthe tumor cells, to a test compound; b) injecting the first set of tumorcells at varying numbers of cells of the first series of host animalsand injecting into a second series of host animals at varying numbers ofcells of the second set of tumor cells; c) comparing tumor formationfrequency and rate in both the first and second series of host animals;and d) determining the effect of the test compound on the number ofsolid tumor stem cells in the first set compared to the second set oftumor cells.
 2. The method of claim 1, wherein the solid tumor ofepithelial origin is selected from the group consisting of coloncarcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostatecancer, squamous cell carcinoma, cervical cancer, lung carcinoma, smallcell lung carcinoma, and bladder carcinoma.
 3. The method of claim 1,wherein the tumor cells are enriched for solid tumor stem cells.
 4. Themethod of claim 1, wherein the cell-surface markers are selected fromthe group consisting of CD44, CD24, and ESA.
 5. The method of claim 1,wherein step d) shows the test compound decreases the number of solidtumor stem cells in the first set of tumor cells by a decrease the tumorformation rate in at least one of the varying cell numbers in the firstseries of host animals compared to the second series of host animals. 6.The method of claim 5, wherein the test compound is an antibody.
 7. Themethod of claim 6, wherein the antibody is a Notch antibody.
 8. Themethod of claim 7, wherein the Notch antibody is an antibody to Notch4.9. A method of diagnosing the effect of a therapeutic compound on solidtumor stem cells from a solid tumor of epithelial origin, the methodcomprising: a) injecting tumor cells from the first tumor sample atvarying numbers of cells into a first series of host animals; b)determining tumor formation frequency and rate in the first series ofhost animals; c) injecting tumor cells from the second tumor sample atvarying numbers of cells into a second series of host animals; d)determining tumor formation frequency and rate in the second series ofhost animals; e) comparing tumor formation rates in the first and secondseries of host animals; and f) diagnosing the effect of the therapeuticcompound to which at least the first tumor sample or the second tumorsample has been exposed.
 10. The method of claim 9, wherein the solidtumor of epithelial origin is selected from the group consisting ofcolon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,prostate cancer, squamous cell carcinoma, cervical cancer, lungcarcinoma, small cell lung carcinoma, and bladder carcinoma.
 11. Themethod of claim 9, wherein the first tumor sample and the second tumorsample are from the same patient.
 12. The method of claim 11, whereinthe first tumor sample is obtained prior to treatment with thetherapeutic compound.
 13. The method of claim 11, wherein the secondtumor sample is obtained after treatment with the therapeutic compoundends.
 14. The method of claim 11, wherein the first tumor sample isexposed to a lower concentration of the therapeutic compound.
 15. Themethod of claim 11, wherein the second tumor sample is exposed to alower concentration of the therapeutic compound.
 16. The method of claim11, wherein the second tumor sample is exposed to the therapeuticcompound in combination with a second therapeutic treatment.
 17. Themethod of claim 9, wherein the first tumor sample and the second tumorsample are from different patients exposed to the therapeutic compound.18. The method of claim 9, wherein the tumor cells are enriched forsolid tumor stem cells.
 19. The method of claim 18, wherein theenrichment is based on cell-surface markers.
 20. The method of claim 19,wherein the cell-surface markers are selected from the group consistingof CD44, CD24, and ESA.